A SNP molecular marker for screening cassava with high potassium content and its application

Abstract

The present invention provides a SNP molecular marker for screening high-potassium content cassava, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, and the base at the 220th position from the 5' end of the sequence shown in SEQ ID No.1 is G or A. The present invention identifies for the first time from a big data level that the SNP located at the 220th position of the nucleotide sequence of SEQ ID NO.1 is closely related to the potassium content of cassava root tubers, and the SNP molecular marker is not limited by the growth stage of cassava, and can be selected in the seedling stage, which greatly reduces the workload of breeding, significantly shortens the cassava breeding selection cycle, improves the breeding efficiency, and can be used in practice for molecular marker-assisted breeding for cassava quality improvement.

Description

Technical Field

[0001] The present invention relates to a SNP marker and application thereof, in particular to a SNP molecular marker for screening cassava with high potassium content and application thereof. Background Art

[0002] Cassava is one of the most important food crops in tropical regions. Known as the "king of starch," it boasts excellent traits such as high yield and tolerance to drought conditions. It is the staple food of nearly one billion people worldwide. While a typical cassava-based diet provides adequate calories, its nutritional value, such as potassium, falls far short of the minimum daily requirement. Therefore, there is an urgent need to genetically modify high-potassium cassava using molecular marker technology.

[0003] Potassium is an essential mineral and electrolyte for the human body, involved in many important physiological functions and maintaining body health and metabolic balance. In hot climates, people, especially those engaged in physical labor, are prone to sweating profusely, which leads to potassium loss. If potassium loss is not replenished in time through diet, it may lead to potassium deficiency. Therefore, there is an urgent need to improve cassava varieties in terms of nutritional fortification, screening or breeding new germplasm or new varieties with significantly increased potassium content to meet the nutritional needs of humans who rely on cassava as their staple food. However, at present, there is no molecular marker information for the high potassium trait in cassava tubers, which seriously limits the selection and breeding of high-potassium cassava varieties.

[0004] SNPs (single nucleotide polymorphisms) primarily refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level, including single base transitions and transversions. By utilizing SNP markers, breeders can screen individuals with superior genotypes at an early stage, accelerating the breeding process and reducing the uncertainty and time costs associated with traditional methods. Furthermore, SNP-assisted selection enables in-depth analysis of the genetic basis of complex traits, thereby improving the precision and efficiency of breeding.

[0005] The present invention selects cassava materials with high potassium content by selecting favorable allele variations of SNP markers, which can significantly improve selection efficiency, shorten the cultivation period of high potassium cassava materials, and accelerate the breeding process. Summary of the Invention

[0006] The present invention aims to overcome the deficiencies in the prior art and provide a SNP molecular marker for screening cassava with high potassium content and its application, which can screen germplasm with high potassium content in tubers at an early stage and promote the breeding process of new varieties of high potassium cassava materials.

[0007] A first aspect of the present invention is to provide a SNP molecular marker for screening cassava with high potassium content. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No. 1, and the base at the 220th position from the 5' end of the sequence shown in SEQ ID No. 1 is G or A (it should be understood that in the sequence listing, the 220th position from the 5' end of SEQ ID No. 1 is represented by "a" only for the normal submission of the nucleotide sequence listing and does not affect the relevant limitations of claim 1 and the description of the present invention).

[0008] The inventors found that the potassium content in the tuberous roots of cassava with the AG genotype at the site of the SNP molecular marker is high.

[0009] Based on the SNP molecular marker, a genotype identification method for potassium content of cassava root tubers can be developed. By analyzing the base sequence of the SNP site, the genotype of the SNP site is AG, which is identified as high-potassium cassava.

[0010] In order to detect the SNP molecular marker, the second aspect of the present invention is to provide a primer pair for detecting the SNP molecular marker described in the first aspect of the present invention, wherein the primer pair is GT1_F: 5'-CAGCCTGCAATTCTTTCTTC-3' and primer GT1_R: 5'-GCCATTGTGTTCTTCAACAAG-3'.

[0011] The third aspect of the present invention is to provide a kit for detecting the SNP molecular marker described in the first aspect of the present invention, which comprises the primer pair described in the second aspect of the present invention.

[0012] The fourth aspect of the present invention is to provide the use of the SNP molecular marker described in the first aspect of the present invention, or the primer pair described in the second aspect of the present invention, or the kit described in the third aspect of the present invention in the early screening of cassava germplasm with high root potassium content.

[0013] A fifth aspect of the present invention provides a method for detecting the potassium content of cassava root tubers, comprising extracting genomic DNA from the cassava to be tested, using the genomic DNA from the cassava to be tested as a template, performing PCR amplification using the primer pair described in the second aspect of the present invention, analyzing the sequence of the PCR amplification product, detecting the SNP molecular marker described in the first aspect of the present invention, determining the genotype of the cassava to be tested, and thereby determining the potassium content of the cassava root tubers to be tested.

[0014] When the genotype of the SNP molecular site is AG genotype cassava, it is preliminarily identified that the potassium content in the cassava root tubers is high.

[0015] A sixth aspect of the present invention provides a cassava breeding method, comprising extracting genomic DNA from each candidate cassava in a candidate cassava population, using the obtained genomic DNA as a template, performing PCR amplification using the primer pair described in the second aspect of the present invention, analyzing the sequence of the PCR amplification product, and detecting the SNP molecular marker described in the first aspect of the present invention to determine the genotype of each candidate cassava, thereby determining the root potassium content trait of each candidate cassava, and breeding the cassava.

[0016] The seventh aspect of the present invention is to provide use of the SNP molecular marker described in the first aspect of the present invention, or the primer pair described in the second aspect of the present invention, or the kit described in the third aspect of the present invention in cassava breeding.

[0017] The present invention, for the first time, identified from big data that the SNP located at position 220 of the nucleotide sequence of SEQ ID NO.1 is closely associated with the potassium content of cassava root tubers. The SNP molecular marker is not restricted by the growth stage of cassava and can be selected during the seedling stage, greatly reducing the breeding workload, significantly shortening the cassava breeding selection cycle, and improving breeding efficiency. It can be used in practice for molecular marker-assisted breeding for cassava quality improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Manhattan plot of SNP markers that are extremely significantly associated with potassium content in cassava roots.

[0019] Figure 2 The diagram shows the Manhattan plot and linkage disequilibrium haplotype block diagram of the local interval of chromosome 17 where the SNP markers that are extremely significantly correlated with the potassium content of cassava root tubers are located, wherein the deepened markers in the interval are the molecular markers screened by the present invention.

[0020] Figure 3 The potassium content differences among different genotypes were compared for SNP markers that were extremely significantly associated with potassium content in cassava roots. The AG allele was significantly associated with high potassium content in roots, ***P<0.001.

[0021] Figure 4 Schematic diagram of different genotypes (AA and AG) of the SNP site of the present invention. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to specific embodiments for a better understanding of the present invention.

[0023] Example 1: Mining SNP sites significantly associated with root potassium content based on genome-wide association analysis (GWAS)

[0024] A total of 297 cassava accessions from China, Brazil, Nigeria, Colombia, and other countries were obtained from the National Cassava Germplasm Resource Center (Danzhou) and sown at the Wenchang base of the Chinese Academy of Tropical Agricultural Sciences, ensuring consistent soil fertility and temperature. Cassava genomic DNA was extracted from young leaf tissue using the Polysaccharide and Polyphenol Plant Genomic DNA Extraction Kit (Tiangen). DNA samples with an OD value between 1.8 and 2.0 and a DNA content of at least 1.5 μg were used for library construction and sequencing. Raw sequencing data were filtered to obtain high-quality data (Q20 >= 90%, Q30 >= 85%) with a GC content between 37% and 39% for subsequent analysis. High-quality sequencing data were aligned to the reference genome using BWA software (v0.7.17, parameters: mem -t 4 -k 32-M). Alignments were sorted and deduplicated using SAMTOOLS (v1.9).

[0025] SNPs (single nucleotide polymorphisms) play a crucial role in breeding, helping breeders accurately identify genetic markers associated with important traits. We used software such as SAMTOOLS to detect SNPs in the sample population. High-quality SNPs were obtained through the following filtering and screening: (1) Q20 quality control (SNPs with a quality value Q20, i.e., sequencing error rate greater than 1%, were filtered out); (2) the support number (coverage depth) of the variant site was >3; (3) the proportion of missing genotypes in the resequencing samples of the variant site was <=20%; and (4) the minimum allele frequency (MAF) of the variant site was >0.05. Finally, ANNOVAR software was used to annotate the population SNPs, obtain the relative position of the variant site to the gene, and detect whether the variant site on the gene is a non-synonymous mutation.

[0026] Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to determine the potassium content of cassava tubers. After drying and pulverizing, the cassava tubers were digested using a wet digestion method. 10 mL of high-grade perchloric acid was slowly added to 100 mL of high-grade nitric acid and mixed thoroughly to prepare a nitric acid-perchloric acid (10+1) mixed solution. 0.5 g of dried cassava tubers were accurately weighed and placed in a glass digestion vessel. 10 mL of the nitric acid-perchloric acid (10+1) mixed solution was added and digested on a hot plate until white smoke appeared and the digestion solution became colorless and transparent or slightly yellow. After cooling, the solution was diluted to 50 mL with ddH2O and mixed thoroughly. A blank test was also performed. The standard working solutions were injected into an inductively coupled plasma optical emission spectrometer (PerkinElmer Optima 8300) to measure the intensity signal response of the analyte lines of the element being measured. A standard curve was plotted with the concentration of the element being measured as the abscissa and the intensity response of the analyte lines as the ordinate. The blank solution and the sample solution were injected into the inductively coupled plasma emission spectrometer respectively, and the signal response value of the analysis line intensity of the element to be measured was measured, and the concentration of the element to be measured in the digestion solution was obtained according to the standard curve.

[0027] Based on genome-wide association studies (GWAS), SNPs significantly associated with root potassium content were identified. GWAS is a method for analyzing the overall association of common genetic variation (single nucleotide polymorphisms and copy number) across the entire genome. This method uses natural populations as the research subject and linkage disequilibrium (LD) between genes (locus) that persists after long-term recombination. By combining the phenotypic diversity of the target trait with the polymorphism of genes (or marker loci), this method can directly identify gene loci or marker loci that are closely associated with phenotypic variation and have specific functions.

[0028] The mixed linear model (MLM) in GEMMA was used to conduct association analysis on the potassium content traits of the population, and the significance of the association was analyzed ( P -value < 0.05) to screen potential candidate SNPs. The results showed that there was a GWAS signal significantly associated with potassium content at the 24.20Mb position of chromosome 17 in cassava (-log10(P-value) = 5.73), and a linkage disequilibrium region of 11 kb was detected, and the candidate gene Sc17g007000 related to cassava potassium content was identified ( Figure 1 , Figure 2). Further analysis revealed that a non-synonymous mutation SNP site Sc17g007000:2924 (A / G) was screened in the 3'UTR of the Sc17g007000 gene, which was significantly associated with the potassium content of cassava roots. It was located at 24,204,846 bp on chromosome 17 (3'UTR of the Sc17g007000 gene). Compared with the AA genotype, the potassium content of cassava roots containing the AG genotype was significantly increased (Table 1, Figure 3 By selecting the AG genotype at this SNP site, cassava materials with high potassium content can be quickly selected or predicted, thereby effectively selecting the potassium content of cassava tubers and accelerating the molecular design breeding process of high-potassium cassava varieties.

[0029] Table 1 Statistics of different genotypes and potassium content of cassava in Sc17g007000:2924

[0030]

[0031] Note: Due to the filtering step, low-quality reads and sites with low coverage are filtered out, so the total number of individuals at each site is ≤297.

[0032] A potassium content survey was conducted on the aforementioned cassava germplasms. Genotypes at the target SNP loci were analyzed for germplasms with particularly high potassium content (greater than or equal to 0.6%). The results showed that among the germplasms with potassium content greater than or equal to 0.6%, 30 of the 248 germplasms had the AA genotype, accounting for 12.10% of the AA genotype, and 16 of the 45 germplasms had the AG genotype, accounting for 35.56% of the AG genotype.

[0033] Example 2: Sc17g007000:2924 SNP marker development

[0034] Based on the cassava SC205 reference genome, a total of 311 bp of upstream and downstream sequences of the SNP site (Sc17g007000:2924 (A / G)) significantly associated with the potassium content of cassava roots were extracted. Primer 5 software was used to design the primer sequence GT1, and PCR amplification was performed using primers GT1_F: 5'-CAGCCTGCAATTCTTTCTTC-3' and GT1_R: 5'-GCCATTGTGTTCTTCAACAAG-3'.

[0035] The PCR reaction system consisted of 1 μl of DNA template, 12.5 μl of PCR mix, 1 μl of forward primer, 1 μl of reverse primer, and 9.5 μl of ddH2O (25 μl total). The PCR protocol was as follows: initial denaturation at 98°C for 5 min, followed by 30 cycles of denaturation at 98°C for 10 sec, annealing at 55°C for 5 sec, and extension at 72°C for 10 sec, followed by extension at 72°C for 7 min.

[0036] The amplified product was recovered and sequenced to obtain a nucleotide sequence containing the above-mentioned SNP (Sc17g007000:2924 (A / G)) site, as shown in SEQ ID No. 1. The SNP site (named Sc17g007000:2924 SNP) is located at the 220 bp site from the 5' end of the sequence shown in SEQ ID NO: 1. The site is represented by "a" in the SEQ ID NO: 1 sequence, and the base at this site is G or A (it should be understood that the 220 bp site from the 5' end of SEQ ID No. 1 in the sequence listing is represented by "a" only for the normal submission of the nucleotide sequence listing and does not affect the relevant limitations of claim 1 and the description of the present invention).

[0037] Example 3 Screening or Detection of High Potassium Cassava Materials

[0038] 78 cassava plants were randomly selected from the cassava planting area of ​​the Wenchang base of the Chinese Academy of Tropical Agricultural Sciences. After sequencing, genotyping and potassium content were investigated. The specific procedures are as follows:

[0039] 1) Extract DNA from the cassava sample to be tested;

[0040] 2) PCR amplification of the GT1 gene was performed using the specific primer pair (primer GT1_F: 5'-CAGCCTGCAATTCTTTCTTC-3' and primer GT1_R: 5'-GCCATTGTGTTCTTCAACAAG-3') to obtain a PCR product. The PCR reaction system was as follows: 1 μl DNA template, 12.5 μl PCR mix, 1 μl forward primer, 1 μl reverse primer, and 9.5 μl ddH2O, for a total of 25 μl. The PCR reaction procedure was as follows: initial denaturation at 98°C for 5 min; 30 cycles of denaturation at 98°C for 10 sec, annealing at 55°C for 5 sec, and extension at 72°C for 10 sec; and extension at 72°C for 7 min.

[0041] 3) The PCR amplification products were detected by 1% agarose gel electrophoresis and then sequenced. The sequencing peaks were then checked to analyze the genotype of the cassava sample. If the 220 bp position of the PCR product showed a single peak (A), the sample was of AA genotype. If the 220 bp position of the PCR product showed a double peak (AG), the sample was of AG genotype. Figure 4 ); the sample to be tested of AG genotype is a cassava material with high potassium content.

[0042] The results of testing 78 cassava samples, as shown in Tables 2 and 3, show that the SNP molecular markers of the present invention have a high accuracy rate for identifying high-potassium cassava. Cassava roots with the AG genotype have substantially higher potassium contents than those with the AA genotype. Specifically, the G allele at the Sc17g007000:2924 SNP locus is highly significantly correlated with potassium content in cassava roots. By selecting cassava materials with the AG genotype at this SNP locus, high-potassium cassava materials can be rapidly selected or predicted, effectively selecting for potassium content in cassava roots and accelerating the molecular design breeding process for high-potassium cassava varieties.

[0043] Table 2 Different genotypes of Sc17g007000:2924 SNP and potassium content (%) in 78 cassava plants

[0044]

[0045] Table 3 Statistics of different genotypes of Sc17g007000:2924 SNP and potassium content in 78 cassava plants

[0046] genotype Number of individuals Average potassium content (%) AA 64 0.454 AG 14 0.568

[0047] While the specific embodiments of the present invention have been described in detail above, these are merely exemplary and the present invention is not limited thereto. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to be encompassed within the scope of the present invention.

Claims

1. Use of a primer pair for screening SNP molecules associated with high potassium content cassava or a kit comprising the primer pair in early screening of cassava germplasm with high potassium content in root tubers, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, and the base at the 220th position from the 5' end of the sequence shown in SEQ ID No.1 is G or A; the potassium content in the tuberous roots of the AG genotype cassava of the SNP molecular marker is high; the primer pair is GT1_F: 5'-CAGCCTGCAATTCTTTCTTC-3' and GT1_R: 5'-GCCATTGTGTTCTTCAACAAG-3'.

2. Use of a primer pair for screening SNP molecules related to high potassium content cassava or a kit comprising the primer pair in the breeding of cassava root tubers for potassium content, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, and the base at the 220th position from the 5' end of the sequence shown in SEQ ID No.1 is G or A; the potassium content in the tuberous roots of the AG genotype cassava of the SNP molecular marker is high; the primer pair is GT1_F: 5'-CAGCCTGCAATTCTTTCTTC-3' and GT1_R: 5'-GCCATTGTGTTCTTCAACAAG-3'.

3. A method for detecting potassium content in cassava root tubers, characterized in that: The genomic DNA of the cassava to be tested is extracted, the genomic DNA of the cassava to be tested is used as a template, a primer pair is used for PCR amplification, the sequence of the PCR amplification product is analyzed, and the SNP molecular marker is detected to determine the genotype of the cassava to be tested, thereby determining the potassium content trait of the tuber of the cassava to be tested, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, and the base at the 220th position from the 5' end of the sequence shown in SEQ ID No.1 is G or A; the AG genotype cassava of the SNP molecular marker has a high potassium content in the tuber; the primer pair is GT1_F: 5'-CAGCCTGCAATTCTTTCTTC-3' and GT1_R: 5'-GCCATTGTGTTCTTCAACAAG-3'.

4. A method for breeding cassava root tubers for potassium content, characterized in that: The genomic DNA of each cassava to be selected in the cassava to be selected population is extracted, the obtained genomic DNA is used as a template, a primer pair is used for PCR amplification, the sequence of the PCR amplification product is analyzed, the SNP molecular marker related to the magnesium content of the cassava root tubers is detected, the genotype of each cassava to be selected is determined, thereby determining the root tuber potassium content trait of each cassava to be selected, and the cassava is bred, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, the base at the 220th position from the 5' end of the sequence shown in SEQ ID No.1 is G or A; the AG genotype cassava of the SNP molecular marker has a high potassium content in the root tubers; the primer pair is GT1_F: 5'-CAGCCTGCAATTCTTTCTTC-3' and GT1_R: 5'-GCCATTGTGTTCTTCAACAAG-3'.

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