Development, detection method and application of SNP molecular markers related to magnesium content in cassava roots

Abstract

The present invention provides a SNP molecular marker related to the magnesium content of cassava root tubers, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, and the base at the 234th position from the 5' end of the sequence shown in SEQ ID No.1 is C or T. The present invention, for the first time, identifies from the big data level that the SNP located at the 234th position of the nucleotide sequence of SEQ ID NO.1 is closely related to the magnesium content of cassava root tubers, and the SNP 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 of cassava nutritional quality.

Description

Technical Field

[0001] The present invention relates to a SNP marker and an application thereof, and in particular to the development, detection method and application of a SNP molecular marker related to magnesium content in cassava root tubers. Background Art

[0002] Cassava (Manihot esculenta) is an important tropical crop, widely grown in tropical and subtropical regions such as Africa, South America and Southeast Asia. As the "king of starch", cassava roots are rich in starch and are the main food source for nearly 1 billion people worldwide. However, although cassava can meet the calorie needs, its content of essential nutrients such as magnesium is far from enough to meet the daily needs of the human body. Therefore, we urgently need to use molecular marker technology to genetically improve cassava to enhance its nutritional value.

[0003] Magnesium is an essential mineral for the human body and is vital to health. Ensuring adequate magnesium intake is very important for maintaining human health and preventing chronic diseases. Therefore, there is an urgent need to improve cassava varieties in terms of nutritional fortification, screen or cultivate new germplasms or new varieties with significantly improved magnesium content to meet the nutritional needs of humans who mainly eat cassava. However, at present, there is no molecular marker information for the high magnesium trait of cassava tubers, which seriously limits the selection of high-magnesium cassava varieties.

[0004] SNP (single nucleotide polymorphism) refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level, including the conversion and transversion of a single base. By using SNP markers, breeders can screen out individuals with excellent genotypes at an early stage, accelerate the breeding process, and reduce the uncertainty and time cost in traditional methods. In addition, SNP-assisted selection makes it possible to conduct in-depth analysis of the genetic basis of complex traits, thereby improving the accuracy and efficiency of breeding.

[0005] The present invention selects cassava materials with high magnesium content by selecting favorable allele variations of SNP markers, which can significantly improve selection efficiency, shorten the cultivation period of high-magnesium 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 development, detection method and application related to the magnesium content of cassava root tubers, which can screen germplasm with high magnesium content in root tubers at an early stage and promote the breeding process of new varieties of high-magnesium cassava materials.

[0007] The first aspect of the present invention is to provide a SNP molecular marker related to the magnesium content of cassava root tubers, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, and the base at the 234th position from the 5' end of the sequence shown in SEQ ID No.1 is C or T (it should be understood that in the sequence list, the 234th position from the 5' end of SEQ ID No.1 is represented by "c" only for the normal submission of the nucleotide sequence list, and does not affect the relevant limitations of claim 1 and the specification of the present invention).

[0008] The inventors found that the magnesium content in the tubers of cassava with CT or CC genotype genotype at the site of the SNP molecular marker is likely to be significantly higher than that of cassava with TT genotype.

[0009] Based on the SNP molecular marker, a genotype identification method for magnesium content in cassava root tubers can be developed. By analyzing the base sequence of the SNP site, the genotype of the SNP site is CT genotype or CC genotype, and the cassava is identified as high-magnesium 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'-CCTGTGCTTGCTACTGTCAAC-3' and primer GT1_R: 5'-GAGCTAGTAGTTGGGACTCTGC-3'.

[0011] The third aspect of the present invention is to provide a kit for detecting the SNP molecular markers 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 as described in the first aspect of the present invention, or the primer pair as described in the second aspect of the present invention, or the kit as described in the third aspect of the present invention in the early screening of cassava germplasm with high magnesium content in root tubers.

[0013] The fifth aspect of the present invention is to provide a method for detecting the magnesium content trait of cassava root tubers: extracting the genomic DNA of the cassava to be tested, using the genomic DNA of the cassava to be tested as a template, using the primer pair described in the second aspect of the present invention to perform PCR amplification, 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 the cassava to be tested, thereby determining the magnesium content trait of the root tubers of the cassava to be tested.

[0014] When the genotype of the SNP molecular site is CT genotype or CC genotype cassava, the cassava is preliminarily identified as a cassava material with a high magnesium content.

[0015] The sixth aspect of the present invention is to provide a cassava breeding method: extracting genomic DNA of each cassava to be selected in a cassava population to be selected, using the obtained genomic DNA as a template, using the primer pair described in the second aspect of the present invention to perform PCR amplification, 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 cassava to be selected, thereby determining the root magnesium content trait of each cassava to be selected, 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 identifies for the first time from a big data level that the SNP at position 234 of the nucleotide sequence of SEQ ID NO.1 is closely associated with the magnesium content of cassava root tubers. 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 breeding workload, significantly shortens the cassava breeding selection cycle, and improves the 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 magnesium content in cassava roots.

[0019] Figure 2 The diagram is a Manhattan plot and a linkage disequilibrium haplotype block plot of a local region of chromosome 10 where a SNP marker extremely significantly correlated with magnesium content in cassava root tubers is located, wherein the deepened markers in the region are the molecular markers screened by the present invention.

[0020] Figure 3 The magnesium content differences among different genotypes of SNP markers that were extremely significantly associated with the magnesium content of cassava roots were compared. The CT and CC alleles were significantly associated with high magnesium content in roots, *P<0.05, **P<0.01, ns means not significant.

[0021] Figure 4 It is a schematic diagram of the typing of different genotypes (TT, CT and CC) of the SNP site of the present invention.

[0022] Specific embodiment

[0023] The present invention will be further described below in conjunction with specific embodiments to provide a better understanding of the present invention.

[0024] Example 1: Mining SNP sites significantly associated with magnesium content in root tubers based on genome-wide association analysis (GWAS)

[0025] 297 cassava samples from China, Brazil, Nigeria, Colombia and other countries were obtained from the National Cassava Germplasm Resource Garden (Danzhou) as research materials and sown in the Wenchang Base of the Chinese Academy of Tropical Agricultural Sciences to ensure consistent planting conditions such as soil fertility and temperature. Young leaf tissues were selected and cassava genomic DNA was extracted using the Polysaccharide and Polyphenol Plant Genomic DNA Extraction Kit (Tiangen). DNA samples with OD values ​​between 1.8 and 2.0 and a content of more than 1.5ug were used to construct libraries for sequencing. The raw sequencing data were filtered to obtain high-quality sequencing data (Q20 >= 90%, Q30 >= 85%) with a GC content between 37% and 39% for subsequent analysis. The high-quality sequencing data were aligned to the reference genome using BWA software (v0.7.17, parameters: mem-t 4-k 32-M), and the alignment results were sorted and deduplicated using SAMTOOLS (v1.9).

[0026] SNP (single nucleotide polymorphism) plays a vital role in breeding and can help breeders accurately identify genetic markers associated with important traits. We used SAMTOOLS and other software to detect population SNPs in samples. High-quality SNPs were obtained through the following filtering and screening: (1) Q20 quality control (filtering out SNPs with a quality value Q20, i.e., sequencing error rate greater than 1%); (2) the support number (coverage depth) of the variant site > 3; (3) the proportion of missing genotypes in the resequencing samples of the variant site <= 20%; (4) the minimum allele frequency (MAF) of the variant site > 0.05. Finally, ANNOVAR software was used to annotate population SNPs, obtain the relative position of the variant site and the gene, and detect whether the variant site on the gene is a non-synonymous mutation.

[0027] Detection of magnesium content in cassava root tubers by inductively coupled plasma optical emission spectrometry (ICP-OES): After drying and crushing the cassava root tubers, the samples were digested by wet digestion. Take 10 mL of high-grade perchloric acid and slowly add it to 100 mL of high-grade nitric acid to mix well, and prepare a nitric acid-perchloric acid (10+1) mixed solution for use. Accurately weigh 0.5 g of dried cassava root tubers in a glass digestion vessel, add 10 mL of nitric acid-perchloric acid (10+1) mixed solution, and digest on a hot plate until white smoke is emitted and the digestion liquid is colorless and transparent or slightly yellow. After cooling, use ddH 2O to 50 mL, mix well and set aside; perform a blank test at the same time. Inject the standard series working solutions into the inductively coupled plasma emission spectrometer (PerkinElmer Optima 8300), measure the intensity signal response value of the analysis line of the element to be measured, and draw a standard curve. Inject the blank solution and the sample solution into the inductively coupled plasma emission spectrometer respectively, measure the signal response value of the intensity of the analysis line of the element to be measured, and obtain the concentration of the element to be measured in the digestion solution according to the standard curve.

[0028] The mixed linear model (MLM) in GEMMA was used to conduct association analysis on the magnesium content traits of the population, and the significance of the association was analyzed by ( P -value < 0.05) to screen potential candidate SNPs. The results showed that there was a GWAS signal significantly associated with magnesium content at the 28.29 Mb position of chromosome 10 of cassava (-log 10 ( P -value) =5.73), and detected a linkage disequilibrium region of 27 kb, and identified the candidate gene Sc10g013360 ( Figure 1 , Figure 2 ). Further analysis revealed that a non-synonymous mutation SNP site Sc10g013360:5497 (C / T) significantly correlated with the magnesium content of cassava roots was screened in the seventh exon of the Sc10g013360 gene, located at 28,284,856 bp on chromosome 10 (the seventh exon of Sc10g013360). Compared with the TT genotype, the magnesium content of cassava roots containing the CT and CC genotypes was significantly increased (Table 1, Figure 3 ).

[0029] Table 1 Statistics of magnesium content in different genotypes of cassava in Sc10g013360:5497

[0030]

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

[0032] The magnesium content of the above materials was investigated, and germplasms with particularly high magnesium content (greater than or equal to 0.18%) were selected for genotype analysis of the target SNP site. The results showed that among the germplasms with magnesium content greater than or equal to 0.18%, there were 27 germplasms with genotype CT, accounting for 23.68% of the CT genotype, 16 germplasms with genotype CC, accounting for 31.37% of the CC genotype, and 18 germplasms with genotype TT, accounting for 7.69% of the TT genotype.

[0033] Example 2 Sc10g013360:5497 SNP marker development

[0034] Based on the cassava SC205 reference genome, a total of 385 bp of upstream and downstream sequences of the SNP site (Sc10g013360:5497 (C / T)) significantly associated with magnesium content in cassava roots were extracted, and the primer sequences were designed using Primer 5 software. PCR amplification was performed using primers GT1_F: 5'-CCTGTGCTTGCTACTGTCAAC-3' and GT1_R: 5'-GAGCTAGTAGTTGGGACTCTGC-3'. The PCR reaction system was: DNA template 1μl, PCR mix 12.5μl, forward primer 1μl, reverse primer 1μl, ddH 2 The PCR reaction program was as follows: 98°C pre-denaturation for 5 min; 98°C denaturation for 10 sec, 55°C annealing for 5 sec, 72°C extension for 10 sec, 30 cycles; 72°C extension for 7 min.

[0035] The amplified product was recovered and sequenced to obtain a nucleotide sequence containing the above-mentioned SNP (Sc10g013360:5497 (C / T)) site, as shown in SEQ ID No. 1. The SNP site (named Sc10g013360:5497 SNP) is located at the 234 bp site from the 5' end of the sequence shown in SEQ ID NO: 1. The site is represented by "c" in the SEQ ID NO: 1 sequence, and the base at this site is C or T (it should be understood that in the sequence table, the 234th site from the 5' end of SEQ ID No. 1 is represented by "c" only for the normal submission of the nucleotide sequence table, and does not affect the relevant limitations of claim 1 and the specification of the present invention).

[0036] Example 3 Screening or Detection of High-Mg Cassava Materials

[0037] 52 cassava plants were randomly selected from the cassava planting area of ​​the Wenchang base of the Chinese Academy of Tropical Agricultural Sciences, and their genotyping and magnesium content were investigated after sequencing. The specific operations are as follows:

[0038] 1) Extract DNA from the cassava sample to be tested;

[0039] 2) PCR amplification was performed with specific primer pair GT1 (primer GT1_F: 5' -CCTGTGCTTGCTACTGTCAAC- 3' and primer GT1_R: 5' -GAGCTAGTAGTTGGGACTCTGC- 3') to obtain PCR products; the PCR reaction system was: DNA template 1μl, PCR mix 12.5μl, forward primer 1μl, reverse primer 1μl, ddH 2 O 9.5μl, a total of 25μl; PCR reaction program: 98℃ pre-denaturation for 5min; 98℃ denaturation for 10sec, 55℃ annealing for 5sec, 72℃ extension for 10sec, 30 cycles; 72℃ extension for 7min;

[0040] 3) The PCR amplification products were sequenced after being detected by 1% agarose gel electrophoresis. The sequencing peak graph was then checked to analyze the genotype of the cassava sample to be tested. When the 234 bp position of the PCR product was a C single peak, the sample to be tested was a CC genotype, when the 234 bp position of the PCR product was a CT double peak, the sample to be tested was a CT genotype, and when the 234 bp position of the PCR product was a TT single peak, the sample to be tested was a TT genotype ( Figure 4 ); then the samples to be tested of CC and CT genotypes are cassava materials with high magnesium content.

[0041] The results are shown in Table 2 and Table 3, which show that the SNP molecular marker of the present invention has a high accuracy rate in identifying cassava with high magnesium content, and the magnesium content in cassava tubers of CC / CT genotype is substantially higher than that in cassava of TT genotype. That is, the C allele of the Sc10g013360:5497 SNP site is extremely significantly correlated with the high magnesium content of cassava. By selecting the C genotype containing the SNP site, cassava materials with high magnesium content can be quickly selected or predicted, and then the magnesium content of cassava tubers can be effectively selected, accelerating the molecular design breeding process of high-magnesium cassava varieties.

[0042] Table 2 Different genotypes of Sc10g013360:5497 SNP and magnesium content (%) in 52 cassava strains

[0043]

[0044] Table 3 Statistics of different genotypes and magnesium content of 48 cassava strains at Sc10g013360:5497 SNP

[0045] genotype Number of individuals Average magnesium content (%) CC 11 0.159 CT 19 0.150 TT 22 0.134

[0046] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Use of a primer pair for detecting SNP molecular markers related to magnesium content in cassava tubers or a kit comprising the primer pair in early screening of cassava germplasm with high magnesium content in tubers, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, the base at the 234th position from the 5' end of the sequence shown in SEQ ID No.1 is C or T, the magnesium content in the root tubers of the CT genotype or CC genotype cassava of the SNP molecular marker is significantly higher than that of the TT genotype cassava, and the primer pair is GT1_F: 5'-CCTGTGCTTGCTACTGTCAAC-3' and GT1_R: 5'-GAGCTAGTAGTTGGGACTCTGC-3'.

2. Use of a primer pair for detecting SNP molecular markers related to magnesium content in cassava tubers or a kit comprising the primer pair in the selection of magnesium content in cassava tubers, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, the base at the 234th position from the 5' end of the sequence shown in SEQ ID No.1 is C or T, the magnesium content in the root tubers of the CT genotype or CC genotype cassava of the SNP molecular marker is significantly higher than that of the TT genotype cassava, and the primer pair is GT1_F: 5'-CCTGTGCTTGCTACTGTCAAC-3' and GT1_R: 5'-GAGCTAGTAGTTGGGACTCTGC-3'.

3. A method for detecting magnesium 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, the SNP molecular marker related to the magnesium content of the cassava tubers is detected, the genotype of the cassava to be tested is determined, and thus the magnesium content trait of the cassava tubers to be tested is determined, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, and the base at the 234th position from the 5' end of the sequence shown in SEQ ID No.1 is C or T; when the genotype of the SNP molecular marker is CT genotype or CC genotype cassava, it is preliminarily identified that the magnesium content in the cassava tubers is high; the primer pair is GT1_F: 5'-CCTGTGCTTGCTACTGTCAAC-3' and GT1_R: 5'-GAGCTAGTAGTTGGGACTCTGC-3'.

4. A method for breeding cassava root tubers for magnesium content, characterized in that: The genomic DNA of each cassava to be selected in the cassava population to be selected 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 tubers is detected, the genotype of each cassava to be selected is determined, thereby determining the magnesium content trait of the tubers 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, and the base at the 234th position from the 5' end of the sequence shown in SEQ ID No.1 is C or T; when the genotype of the SNP molecular marker is CT genotype or CC genotype cassava, it is preliminarily identified that the magnesium content in the tubers of cassava is high; the primer pair is GT1_F: 5'-CCTGTGCTTGCTACTGTCAAC-3' and GT1_R: 5'-GAGCTAGTAGTTGGGACTCTGC-3'.

Images