A snp marker related to branch number of sweet potato base part and application thereof
By using SNP markers developed at position 23703203 on sweet potato chromosome 12 and KASP technology, the problem of lack of effective markers in sweet potato breeding was solved, enabling rapid and accurate identification of basal branch numbers and improving breeding efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XUZHOU INST OF AGRI SCI IN JIANGSU XUHUAI DISTRICT (JIANGSU XUZHOU SWEETPOTATO CENT)
- Filing Date
- 2025-04-09
- Publication Date
- 2026-05-01
AI Technical Summary
In existing technologies, there is a lack of effective molecular markers in sweet potato breeding for screening and identifying the number of branches of the aboveground parts, resulting in low breeding efficiency.
A SNP marker (Chr12_23703203) located at position 23703203 on chromosome 12 of sweet potato was developed. Primers were designed using KASP technology for genotyping, and FAM and VIC fluorescent tags were used to distinguish between three genotypes: CC, TT, and TC, achieving rapid and accurate identification of the number of basal branches.
This technology enables rapid and accurate identification of the number of basal branches in sweet potatoes at the DNA level, shortening the trait screening cycle, improving breeding efficiency, and allowing for early screening of sweet potato varieties with multiple basal branches, thus advancing the breeding process.
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Figure CN120158548B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular breeding, specifically relating to an SNP marker related to the number of basal branches in sweet potato and its application. Background Technology
[0002] Sweet potato [Ipomoea batatas (L.) Lam.] is a versatile hexaploid crop, used as food, feed, industrial raw material, and a new energy source. The yield and quality traits of the underground tubers are closely related to the growth of the above-ground parts, exhibiting both mutual promotion and restriction. Studies have shown that basal branching is an important reference for high-yielding variety selection. In sweet potato breeding, varieties with more basal branches and relatively shorter vine lengths are beneficial for field management and harvesting, and also for increasing tuber yield. Therefore, further research on the number of basal branches in sweet potato is of great significance for revealing the genetic patterns of important traits and improving the efficiency of sweet potato variety selection.
[0003] The number of basal branches in sweet potato is a quantitative trait controlled by multiple genes, and its phenotype is the result of the interaction between genotype and environment. With the development of molecular biology, constructing molecular linkage maps of sweet potato based on molecular marker technology to locate quantitative trait loci (QTLs) related to certain traits has become an important direction in sweet potato molecular breeding. In recent years, QTL mapping studies in sweet potato have mainly focused on quality traits such as starch content, dry matter content, and β-carotene content. The lack of major genes and molecular markers for identifying aboveground phenotypic traits in sweet potato, and the scarcity of gene resources that can be used for breeding of aboveground multi-basal branching traits, remains a major problem facing current sweet potato breeding work. Summary of the Invention
[0004] The purpose of this invention is to provide a KASP molecular marker related to the number of basal branches in sweet potato.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides an application of a reagent for detecting SNP markers in sweet potato breeding. The SNP marker is Chr12_23703203, located at position 23703203 on sweet potato chromosome 12, and has a base polymorphism of T / C.
[0007] Preferably, the SNP marker Chr12_23703203 is associated with the number of basal branches of sweet potato;
[0008] The base polymorphism T / C has three genotypes: CC, TT, and TC.
[0009] Among them, the CC genotype is the homozygous type with the SNP site being C, which is characterized by a high number of branches in the multibasal branch, with the number of branches being greater than or equal to 25;
[0010] The TT genotype is the homozygous type of the SNP locus being T, characterized by a low number of basal branches, with the number of basal branches being less than or equal to 15.
[0011] The present invention provides an SNP-KASP marker containing the SNP marker, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0012] This invention provides a primer set for detecting the SNP-KASP marker, consisting of upstream primer A and / or upstream primer B and downstream primer C;
[0013] The upstream primer A is shown in SEQ ID NO.2:
[0014] The upstream primer B is shown in SEQ ID NO.3:
[0015] The downstream primer C is shown in SEQ ID NO.4.
[0016] Preferably, a FAM fluorescent tag is added to the 5' end of the upstream primer A;
[0017] A VIC fluorescent tag was added to the 5' end of the downstream primer B.
[0018] The present invention provides a kit for detecting the SNP-KASP marker, comprising the above-described primer set.
[0019] This invention provides the application of the SNP-KASP marker, the primer set, or the kit in sweet potato breeding.
[0020] Preferably, the application of sweet potato breeding refers to its application in screening or identifying the branching number trait of sweet potatoes with multiple basal branches.
[0021] The present invention also provides a method for screening or identifying the multibasal branching trait of sweet potato, the steps of which are: using the genomic DNA of the leaves of the sweet potato to be tested as a template, performing KASP with the primer set, and performing genotyping based on the fluorescence signal. The genotype is CC, which indicates that the sweet potato has the multibasal branching trait.
[0022] Preferably, when the fluorescence signal is red, the genotype of the sweet potato to be tested is CC;
[0023] When the fluorescence signal is blue, the genotype of the sweet potato to be tested is TT;
[0024] When the fluorescence signal is green, the genotype of the sweet potato is TC.
[0025] This invention discloses a set of SNP markers and SNP-KASP markers related to the number of basal branches in sweet potato, which can be used to identify the number of basal branches in sweet potato. The SNP marker is located at position 23703203 on sweet potato chromosome 12, with a base polymorphism of T / C, named Chr12_23703203(T / C). The SNP-KASP marker includes the aforementioned SNP locus. The markers provided by this invention can achieve the evaluation of the number of basal branches in known sweet potato varieties and the screening of sweet potato germplasm resources with multiple branches at the DNA level. The typing and identification method provided by this invention is simple and easy to operate, requires no treatment of sweet potatoes, and can be performed at any stage of the sweet potato growth period, greatly shortening the screening cycle of aboveground traits, improving breeding efficiency, and improving the number of basal branches in sweet potato.
[0026] This invention can be used for the rapid and accurate identification or auxiliary identification of the basal branching number trait in sweet potatoes, enabling early screening of sweet potato materials and its application in marker-assisted breeding. Applying the substances of this invention to marker-assisted selection of the basal branching number trait in sweet potatoes can rapidly screen out sweet potato varieties (germplasm) with multiple basal branching numbers, thereby accelerating the process of breeding aboveground parts of sweet potatoes. This has significant theoretical and economic value in the research of exploring sweet potato germplasm resources and breeding sweet potato varieties with multiple basal branching numbers. Attached Figure Description
[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0028] Figure 1 The results of sweet potato natural population typing based on Chr12_23703203 markers in Example 1;
[0029] Figure 2 The genotyping results of Chr12_23703203 in 52 sweet potato varieties. Detailed Implementation
[0030] During the application of primer sets, the primer sets may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive markers, such as 32P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The marker can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, optionally, charge-neutral. The marker can include nucleic acid sequences or protein sequences or combinations thereof, provided that the sequence containing the marker is detectable. In some embodiments, nucleic acids are detected directly without a marker (e.g., direct sequence reading).
[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0032] Example 1
[0033] 1.1 Discovery of SNP markers for basal branching genes in sweet potato
[0034] The 214 natural sweet potato populations used for GWAS analysis and QTL mapping were planted in Xuzhou, Jiangsu Province, from mid-June 2023 to 2024. A completely randomized block design was used with 3 replicates, 3 rows, 2m row length, 90cm row spacing, and 10 plants per row. The number of basal branches was investigated 90 days after planting. Six individual plants were randomly selected from each row, and the number of basal branches was recorded.
[0035] Genomic DNA was extracted from young leaves of sweet potato plants. The DNA concentration was determined using a NanoDrop2000c spectrophotometer. The DNA samples were adjusted to a standard concentration of 50 ng / μL, and the DNA quality was then tested using a 0.8% agarose gel. DNA samples that passed the quality test were then used for SNP mining.
[0036] 1.2 Convert SNP markers to SNP-KASP markers and design primer sets for detecting these markers.
[0037] Sequences from 214 samples were downloaded from the NCBI database (accession number PRJNA857483). Quality control of genotyping results was performed using PLINK software. SNPs with minor allele frequencies (MAF) > 0.05 and missing data < 5% were selected for genome-wide association studies (GWAS). GWAS analysis was performed using a mixed linear model (CMLM). The threshold for significant p-values was set to 1.0e-5 by default. Multiple SNPs that can be stably detected under various conditions were identified. One SNP, located at position 23703203 on sweet potato chromosome 12, was named Chr12_23703203. After conversion to the SNP-KASP marker, it was named KASP_Chr12_23703203. Its nucleotide sequence (90 bp) is (SEQ ID NO.1): 5'-GAGAGGATGGAACTTATCTACATTTG[T / C]ACATTCTTATCATCTAA TATAGTGGTGGGCTACTGGGCTCACAGTGTTGGCAAGTCTTAAGTT-3'
[0038] The nucleotide Y at position 27 of the KASP_Chr12_23703203 sequence (Y being either T or C) is the selected SNP site Chr12_23703203, which is a polymorphism of T or C. The genotypes of this SNP site are of three types: CC, TT, or TC. The CC genotype is homozygous for the SNP site being C, the TT genotype is homozygous for the SNP site being T, and the TC genotype is heterozygous for both T and C at the SNP site. The genotyping results of 214 natural sweet potato populations are as follows: Figure 1 As shown.
[0039] A primer set for detecting SNP-KASP markers based on KASP technology was designed, referred to 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-labeled primer working solution:
[0043] KASP primers were designed based on KASP_Chr12_23703203, and the polymorphism of this SNP site is a T / C base difference. The target sequence containing the SNP site is shown in SEQ ID NO.1;
[0044] Primer FAM-A is a primer (SEQ ID NO.5) with the FAM fluorescent tag sequence added to the 5' end of upstream primer A, and primer C amplifies the fragment where KASP_Chr12_23703203 is C. The fluorescent signal (red light) of the group can be read using an ELISA reader or a real-time PCR instrument.
[0045] Primer VIC-B is a primer (SEQ ID NO.6) with the VIC fluorescent tag sequence added to the 5' end of the downstream primer B. It amplifies KASP_Chr12_23703203 as a T fragment with primer C. The fluorescent signal (blue light) of the FAM group can be read by an ELISA reader or a real-time PCR instrument.
[0046] The hybrid TC exhibits green fluorescence.
[0047] Example 2
[0048] The experimental materials consisted of 214 sweet potato varieties, which were planted in Xuzhou, Jiangsu Province in mid-June of 2023 and 2024. A completely randomized block design was used with 3 replicates, 3 rows, 2m row length, 90cm row spacing, and 10 plants per row. The number of basal branches was investigated 90 days after planting. From these, 52 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 tagged using Chr12_23703203, and the specific method is as follows;
[0050] (1) PCR amplification system and procedure. Genomic DNA was extracted from sweet potato leaves and dissolved in 200 μl TE. The DNA quality was determined by 1% agarose gel electrophoresis. The extracted DNA was required to be free of obvious impurities, with clear bands and no degradation. After DNA concentration was determined, it was uniformly diluted to 50 ng / μl. Using the diluted sweet potato genomic DNA as a template, PCR amplification was performed. 12 μl each of upstream primer A (100 μM) and upstream primer B (100 μM) and 30 μl of downstream primer C (100 μM) were taken from each primer and made up to 100 μl with sterile ultrapure water. This was used as the KASP-labeled primer working solution and stored at 4℃ for later use.
[0051] The PCR amplification system consisted of: 1.5 μl template DNA, 0.0417 μl primer working solution, 0.75 μl 2×KASP Master Mix (LGC, product number: KBS-1050-112), and sterile ultrapure water to a final volume of 3 μl.
[0052] The PCR reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s and annealing for 20 s (the first annealing temperature was 61℃, and the temperature was reduced by 0.6℃ for each cycle) for a total of 10 cycles; 94℃ denaturation for 20 s and 55℃ annealing for 1 min for a total of 26 cycles; 72℃ extension for 3 min and storage at 4℃.
[0053] (2) Genotyping.
[0054] After the PCR reaction, a fluorescence signal reader (Omega, BMGLABTECH, Germany) and a fluorescence detection system (Araya) were used to convert the fluorescence signals into analyzable numerical values for fluorescence data reading of the reaction products. The fluorescence scanning results were graphically displayed using the ggplot package in R software. C-base types exhibited FAM fluorescence, distributed near the x-axis; T-base types exhibited VIC fluorescence, distributed near the y-axis; samples with no detected signal were distributed near the origin.
[0055] The fluorescence plate was read using an ABI 7500 real-time PCR instrument at 35°C for 30 seconds. Genotyping was performed by reading the fluorescence values at the terminal ends. If a blue fluorescence signal was observed, the genotype of the tested sweet potato KASP_Chr12_23703203 was TT (i.e., the Chr12_23703203 in the sweet potato genome is homozygous for T); if a red fluorescence signal was observed, the genotype was CC (i.e., the Chr12_23703203 in the sweet potato genome is homozygous for C); and if a green fluorescence signal was observed, the genotype was TC (i.e., the Chr12_23703203 in the sweet potato genome is heterozygous for both T and C). The results are as follows: Figure 2 As shown in Table 1.
[0056] Table 152 results of genotyping and number of basal branches for sweet potato varieties.
[0057]
[0058]
[0059] Depend on Figure 2As shown in Table 1, among the 52 sweet potato varieties, 28 varieties showed the TT genotype (blue), 23 varieties showed the TC genotype (green), and 1 variety showed the CC genotype (red). Black represents the negative control. The T-test statistical test showed that the gene effect of KASP_Chr12_23703203 was significantly different (P<0.05). The CC genotype sweet potato had a significantly higher number of basal branches than the TT genotype. The TT genotype varieties mostly had fewer than 15 branches, while the TC genotype sweet potato varieties, although having a higher average number of branches than the TT genotype, showed inconsistent branching.
[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. The application of a reagent for detecting SNP sites in identifying the number of basal branches in sweet potato, characterized in that, The SNP site is Chr12_23703203, located at position 23703203 on chromosome 12 of sweet potato, and the base polymorphism is T / C; The upstream and downstream nucleotide sequences of the SNP site are shown in SEQ ID NO.1, and the SNP site is located at the 27th nucleotide of the nucleotide sequence shown in SEQ ID NO.
1. The NCBI accession number for the sweet potato reference sequence used in screening for the SNPs is PRJNA857483.
2. The application as described in claim 1, characterized in that, The SNP site has a base polymorphism T / C, with three genotypes: CC, TT, and TC. Among them, the CC genotype is the homozygous type with the SNP site being C, which is characterized by a high number of branches in the multi-base position; The TT genotype is the homozygous type with the SNP locus being T, and it is characterized by a low number of basal branches.
3. The application of an SNP-KASP marker related to the number of basal branches in sweet potato breeding, characterized in that, The nucleotide sequence of the SNP-KASP marker is shown in SEQ ID NO.1, and its nucleotide at position 27 is a polymorphism of T or C.
4. The application as described in claim 3, characterized in that, The application of sweet potato breeding refers to its use in screening or identifying the branching number trait of sweet potatoes with multiple basal branches.
5. A method for screening or identifying the branch number trait of multibasal branches in sweet potato, characterized in that, Includes the following steps: Using the leaf genomic DNA of the sweet potato to be tested as a template, KASP was performed using the KASP primer set, and genotyping was performed based on the fluorescence signal. The genotype was CC, which means that the sweet potato has the trait of multiple basal branching. The KASP primer set consists of upstream primer A and upstream primer B and 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 in SEQ ID NO.4; When performing KASP, a FAM fluorescent tag must be added to the 5' end of the upstream primer A; A VIC fluorescent tag should be added to the 5' end of the upstream primer B.
6. The method as described in claim 5, characterized in that, When the fluorescence signal appears red, the genotype of the sweet potato to be tested is CC; When the fluorescence signal is blue, the genotype of the sweet potato to be tested is TT; When the fluorescence signal is green, the genotype of the sweet potato to be tested is TC.