A SNP molecular marker related to calcium content in cassava root tubers and its application

Abstract

The present invention provides a SNP molecular marker related to the calcium 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 299th 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 299th position of the nucleotide sequence of SEQ ID NO.1 is closely related to the calcium content of cassava root tubers, 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 invention relates to a SNP marker and an application thereof, and in particular to a SNP molecular marker related to the calcium content of cassava root tubers and an application thereof. Background Art

[0002] Cassava is one of the most important food crops in tropical regions. It has excellent traits such as high yield and resistance to barrenness. It is known as the "king of starch" and is the staple food of nearly 1 billion people in the world. Although a typical diet based on cassava can provide enough calories, the nutrients in it, such as calcium, are less than 30% of the minimum daily requirement of the human body. Calcium is an essential mineral for maintaining the normal functioning of various functions of the body. Therefore, it is urgent to improve cassava varieties in terms of nutritional fortification, screen or cultivate new germplasm or new varieties with significantly increased calcium content, so as to meet the nutritional needs of humans who mainly eat cassava. However, at present, there is no molecular marker information for the high calcium trait of cassava tubers, which seriously limits the variety selection of high calcium cassava. Therefore, it is urgent to use molecular marker technology for genetic improvement of high calcium cassava.

[0003] The present invention selects cassava materials with high calcium content by selecting favorable allele variations of SNP markers, which can significantly improve selection efficiency, shorten the cultivation period of high calcium cassava materials, and accelerate the breeding process. Summary of the invention

[0004] The present invention aims to overcome the deficiencies in the prior art and provide a SNP molecular marker related to the calcium content of cassava roots and its application, which can screen germplasm with high calcium content in roots at an early stage and promote the breeding process of new varieties of high-calcium cassava materials.

[0005] The first aspect of the present invention is to provide a SNP molecular marker related to the calcium 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 299th 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 table, the 299th position from the 5' end of SEQ ID No.1 is represented by "a" 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).

[0006] The inventors found that the calcium content in the tubers of cassava with AA or AG genotype at the site of the SNP molecular marker is likely to be significantly higher than that of cassava with GG genotype.

[0007] Based on the SNP molecular marker, a genotype identification method for calcium content of cassava root tubers can be developed. By analyzing the base sequence of the SNP site, the genotype of the SNP site is AA or AG, which is identified as high-calcium cassava.

[0008] 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'-CATAATTCTCACCTCGTCGC-3' and primer GT1_R: 5'-CGGTTATGAATAGGGACAAGAG-3'.

[0009] 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.

[0010] 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 calcium content in root tubers.

[0011] The fifth aspect of the present invention is to provide a method for detecting the calcium content of cassava root tubers: extracting 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 calcium content trait of the cassava root tubers to be tested.

[0012] When the genotype of the SNP molecular locus is AA genotype or AG genotype cassava, it is preliminarily identified that the calcium content in the cassava root tubers is high.

[0013] 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 calcium content trait of each cassava to be selected, and breeding the cassava.

[0014] 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.

[0015] The present invention identifies for the first time from a big data level that the SNP at position 299 of the nucleotide sequence of SEQ ID NO.1 is closely associated with the calcium 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

[0016] Figure 1 Manhattan plot of SNP markers that are extremely significantly associated with calcium content in cassava roots.

[0017] Figure 2 The diagram is a Manhattan plot and a linkage disequilibrium haplotype block diagram of a local interval of chromosome 1 where a SNP marker extremely significantly correlated with the calcium content of cassava root tubers is located, wherein the deepened markers within the interval are the molecular markers screened by the present invention.

[0018] Figure 3 The calcium content difference between different genotypes of the SNP markers extremely significantly associated with the calcium content of cassava roots of the present invention was compared. The AA / AG allele was significantly associated with high calcium content in the roots, *** means P<0.001, and ns means not significant.

[0019] Figure 4 It is a schematic diagram of typing different genotypes (AA, AG and GG) of the SNP site of the present invention. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with specific embodiments to provide a better understanding of the present invention.

[0021] Example 1: Mining SNP sites significantly associated with root calcium content based on genome-wide association analysis (GWAS)

[0022] 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).

[0023] SNP (single nucleotide polymorphism) mainly 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. We used software such as SAMTOOLS 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.

[0024] Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to detect the calcium content of cassava root tubers: After the cassava root tubers were crushed and dried, 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 and mix well to 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 solution is colorless and transparent or slightly yellow. After cooling, dilute to 50 mL with ddH2O and mix well for use; at the same time, a blank test was performed. The standard series working solutions were injected into an inductively coupled plasma optical emission spectrometer (PerkinElmer Optima 8300), and the intensity signal response value of the analysis line of the element to be measured was determined to draw a standard curve. The blank solution and the sample solution were respectively injected into the inductively coupled plasma emission spectrometer, 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.

[0025] The mixed linear model (MLM) in GEMMA was used to conduct association analysis on the calcium 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 calcium content at the 30.05Mb position of cassava chromosome 1 (-log 10 ( P -value) =10.61), and detected a linkage disequilibrium region of 40 kb, and identified the candidate gene Sc01g011640 related to the calcium content of cassava ( Figure 1 , Figure 2 ). Further analysis revealed that a non-synonymous mutation SNP site Sc01g011640:373 (A / G) significantly correlated with the calcium content of cassava roots was screened in the second exon of the Sc01g011640 gene, located at 30,026,095 bp on chromosome 1. Compared with the GG genotype, the calcium content of cassava roots containing the AA and AG genotypes was significantly increased (Table 1, Figure 3 ).

[0026] Table 1 Statistics of different genotypes and calcium content of cassava in Sc01g011640:373

[0027]

[0028] 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.

[0029] The calcium content of the cassava germplasms tested above was counted, and the genotypes of the target SNP sites were counted for germplasms with particularly high calcium content (greater than or equal to 0.2%). The results showed that among the germplasms with calcium content greater than or equal to 0.2%, there were 20 germplasms with AA genotype, accounting for 33.9% of the AA genotype; 45 germplasms with AG genotype, accounting for 40.54% of the AG genotype; and 18 germplasms with GG genotype, accounting for 15.52% of the GG genotype.

[0030] Example 2 Sc01g011640:373 SNP marker development

[0031] Based on the cassava SC205 reference genome, a total of 449 bp of upstream and downstream sequences of the SNP site (Sc01g011640:373 (A / G)) significantly associated with the calcium content of cassava roots were extracted. Primer sequences were designed using Primer 5 software, and PCR amplification was performed using primers GT1_F: 5'-CATAATTCTCACCTCGTCGC-3' and GT1_R: 5'-CGGTTATGAATAGGGACAAGAG-3'. The PCR reaction system was: DNA template 1μl, PCR mix 12.5μl, forward primer 1μl, reverse primer 1μl, ddH2O 9.5μl, a total of 25μl. The PCR reaction program was: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 30 sec, 55℃ annealing for 15 sec, 72℃ extension for 30 sec, 30 cycles; 72℃ extension for 7 min.

[0032] The amplified product was recovered and sequenced to obtain a nucleotide sequence containing the above-mentioned SNP (Sc01g011640:373 (A / G)) site, as shown in SEQ ID No. 1. The SNP site (named Sc01g011640:373 SNP) is located at the 299 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 in the sequence table, the 299th site from the 5' end of SEQ ID No. 1 is represented by "a" 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).

[0033] Example 3 Screening or Detection of High-Calcium Cassava Materials

[0034] 66 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 calcium content were investigated after sequencing. The specific operations are as follows:

[0035] 1) Extract DNA from the cassava sample to be tested;

[0036] 2) PCR amplification was performed with specific primer pair GT1 (primer GT1_F: 5' -CATAATTCTCACCTCGTCGC- 3' and primer GT1_R: 5' -CGGTTATGAATAGGGACAAGAG- 3') to obtain PCR products; the PCR reaction system was: DNA template 1μl, PCR mix 12.5μl, forward primer 1μl, reverse primer 1μl, ddH2O 9.5μl, a total of 25μl; the PCR reaction program was: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 30 sec, 55℃ annealing for 15 sec, 72℃ extension for 30 sec, 30 cycles; 72℃ extension for 7 min;

[0037] 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 299 bp position of the PCR product was a single peak of A, the sample to be tested was of the AA genotype; when the 299 bp position of the PCR product was a double peak of AG, the sample to be tested was of the AG genotype; when the 299 bp position of the PCR product was a single peak of G, the sample to be tested was of the GG genotype ( Figure 4 ); the sample to be tested of AA / AG genotype is a cassava material with high calcium content.

[0038] 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 calcium content, and the calcium content in cassava tubers of AA / AG genotype is substantially higher than that of cassava of GG genotype. That is, the A allele of the Sc01g011640:373 SNP site is extremely significantly correlated with the high calcium content of cassava. By selecting the A genotype of the SNP site, cassava materials with high calcium content can be quickly selected or predicted, and then the calcium content of cassava tubers can be effectively selected, accelerating the molecular design breeding process of high calcium cassava varieties.

[0039] Table 2 Different genotypes of Sc01g011640:373 SNP and calcium content (%) in 48 cassava strains

[0040]

[0041] Table 3 Statistics of different genotypes and calcium content of 48 cassava strains in Sc01g011640:373

[0042] genotype Number of individuals Average calcium content (%) AA 17 0.210 AG 25 0.202 GG 24 0.142

[0043] 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 calcium content in cassava tubers or a kit comprising the primer pair in early screening of cassava germplasm with high calcium content in tubers, It is characterized in that The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No. 1, the base at the 299th position from the 5' end of the sequence shown in SEQ ID No. 1 is G or A, and the calcium content in the root tubers of cassava with AA genotype or AG genotype of the SNP molecular marker is significantly higher than that of cassava with GG genotype.

2. Use of a primer pair for detecting SNP molecular markers related to calcium content in cassava tubers or a kit comprising the primer pair in the selection of calcium content in cassava tubers, It is characterized in that The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No. 1, the base at the 299th position from the 5' end of the sequence shown in SEQ ID No. 1 is G or A, and the calcium content in the root tubers of cassava with AA genotype or AG genotype of the SNP molecular marker is significantly higher than that of cassava with GG genotype.

3. A method for detecting the calcium content of cassava root tubers, It is 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 related to the calcium content of the cassava tubers is detected to determine the genotype of the cassava to be tested, thereby determining the calcium content trait of the cassava tubers to be tested, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, the base at the 299th position from the 5' end of the sequence shown in SEQ ID No.1 is G or A, and when the genotype of the SNP molecular marker is AA genotype or AG genotype cassava, it is preliminarily identified that the calcium content in the cassava tubers is high.

4. A method for breeding cassava root tubers for calcium content, It is 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, and a SNP molecular marker related to the calcium content of the cassava root tubers is detected to determine the genotype of each cassava to be selected, thereby determining the root tuber calcium content trait of each cassava to be selected, and breeding the cassava; the nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.1, the base at the 299th position from the 5' end of the sequence shown in SEQ ID No.1 is G or A, and when the genotype of the SNP molecular marker is AA genotype or AG genotype cassava, it is preliminarily identified that the calcium content in the cassava root tubers is high.

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