CN116162725A - SNP marker related to color and anthocyanin content of cassava root endothelium and application thereof - Google Patents
SNP marker related to color and anthocyanin content of cassava root endothelium and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP marker related to the color and anthocyanin content of the inner skin of cassava tubers and application thereof, wherein the nucleotide sequence of the SNP marker is shown as SEQ ID No. 1; a SNP locus C/T is arranged at 262bp of the nucleotide sequence of the SNP marker. Firstly, extracting DNA of a cassava sample to be detected; carrying out PCR amplification by using a specific primer pair rc1 to obtain a PCR product; and checking a sequencing peak diagram, analyzing the genotype of the cassava sample to be detected, and screening to obtain the cassava material with high anthocyanin content and red root endothelia. The method can realize early prediction and rapid screening of the color and anthocyanin content of the inner skin of the cassava tubers, accelerate the breeding process of improving the appearance and the nutrition quality of the cassava, and have important scientific and economic values.
Description
Technical Field
The invention relates to the technical field of biological breeding, in particular to an SNP marker related to the color of the inner skin of cassava tubers and anthocyanin content and application thereof
Background
Anthocyanin is a water-soluble natural pigment widely existing in plant petals, fruits and other parts in the nature, and plays an important role in improving the color value of fruits and vegetables, strengthening food nutrition, maintaining human health and the like. Cassava (Manihot esculenta Crantz) is a perennial root crop of the genus cassava of the family Euphorbiaceae, and provides a food source for about six hundred million people worldwide, and plays a very important role in agricultural and industrial production in China. The starch content of the cassava tubers is high, but the content of bioactive substances such as anthocyanin and the like is low, so that the appearance and the nutritional quality of the cassava tubers are seriously influenced.
Compared with white cassava tubers, the yellow, pink and purple cassava tubers have the advantages that the expression of anthocyanin biosynthesis genes such as CHI, F3H, F, 3'5' H and DFR (regulated by MYB transcription factors) is up-regulated, the anthocyanin content is obviously increased, and the tuber color is bright and popular with consumers, so that the purple cassava tubers have higher appearance and nutritional value. The molecular markers related to the color and anthocyanin content of tuberous roots are excavated, which has important significance for screening or cultivating cassava materials with high Yan Zhi and high anthocyanin content, and is a difficult problem to be solved in the current cassava biological breeding field.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides an SNP marker related to the color and anthocyanin content of the inner skin of the cassava tuberous root and application thereof, can provide important basis for screening the cassava material with high anthocyanin content, cultivating the cassava material with high Yan Zhi and high nutrition quality, and quickens the breeding process.
In order to achieve the purpose, the invention designs an SNP marker related to the color and anthocyanin content of the inner skin of the cassava root tuber, wherein the nucleotide sequence of the SNP marker is shown as SEQ ID No. 1; a SNP locus C/T is arranged at 262bp of the nucleotide sequence of the SNP marker; this site was designated as the Chr2:6158256 (SNP2_ 6158256) site.
The invention also provides a specific primer pair for obtaining the SNP marker, wherein the primer pair rc1 is as follows:
primer rc1_f:5'-TCTTACAATGGGATAAATTAGTGTTATTA-3' the number of the individual pieces of the plastic,
primer rc1_r:5'-TTGGGTAGTTAGGTAGATGGGTTT-3'.
The invention also provides a method for obtaining the SNP marker, which uses cassava genome DNA as a template and the following primer pair for PCR amplification;
primer rc1_f:5'-TCTTACAATGGGATAAATTAGTGTTATTA-3' the number of the individual pieces of the plastic,
primer rc1_r:5'-TTGGGTAGTTAGGTAGATGGGTTT-3';
obtaining SNP marker, the nucleotide sequence of which is shown as SEQ ID No. 1.
The invention also provides a kit for screening or detecting cassava materials with high anthocyanin content and red root endothelia, which contains the specific primer pair rc1 of claim 2.
The invention also provides application of the kit in screening or detecting cassava materials with high anthocyanin content and red root endothelia.
The invention also provides a use method of the kit, which comprises the following steps:
1) Extracting DNA of a cassava sample to be detected;
2) Carrying out PCR amplification by using a specific primer pair rc1 to obtain a PCR product;
3) The PCR amplification products were detected by 1% agarose gel electrophoresis and then sequenced. And then checking a sequencing peak diagram, analyzing the genotype of the cassava sample to be tested, and screening to obtain the cassava material with high anthocyanin content and red root endothelia.
Preferably, in the step 2),
the PCR reaction system is as follows: DNA template 2. Mu.l, PCR mix 25. Mu.l, primer rc1_F2mu.l, primer rc1_R2mu.l, d 2 H 2 O19. Mu.l, total 50. Mu.l;
the PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 53℃for 30sec, elongation at 72℃for 30sec,38 cycles; extending at 72℃for 7min.
Preferably, in the step 3), the comparison result according to the sequencing is as follows:
when the 262bp position of the PCR product is T unimodal, the sample to be detected is TT genotype,
when the 262bp position of the PCR product is CT double peak, the sample to be detected is CT genotype,
when the 262bp position of the PCR product is C single peak, the sample to be detected is CC genotype; the sample to be tested of the CC genotype is cassava material with high anthocyanin content and red root endothelia.
The principle of the invention is as follows:
whole genome association analysis (GWAS) is an important method for identifying the link between phenotype and genotype (marker) in natural populations based on linkage disequilibrium, and is an effective means of mining excellent alleles and developing molecular markers. The single nucleotide polymorphism (Single Nucleotide Polymorphisms, SNP) refers to the change of DNA sequence caused by single base variation, and has the characteristics of large quantity, wide distribution, large-scale detection and the like. And the SNP markers covering the whole genome are adopted to carry out association analysis, genes related to the color and anthocyanin content of the cassava tuberous root are mined, and molecular markers are developed, so that the creation of the cassava material with high Yan Zhi and high anthocyanin content can be quickened.
The invention has the beneficial effects that:
(1) The invention creatively provides an SNP marker related to the color and anthocyanin content of the inner skin of the cassava tubers, and proves the correlation between different genotypes and the color and anthocyanin content of the inner skin of the cassava tubers;
(2) By detecting the SNP marker, the early prediction and quick screening of the color and anthocyanin content of the inner skin of the cassava tubers can be realized, the breeding process of improving the appearance and nutrition quality of the cassava is quickened, and the method has important scientific value and economic value.
Drawings
FIG. 1 is a graph of a genome-wide association analysis of the color of the endothelium of cassava tubers (SR);
in the figure, FIG. 1A is a Manhattan diagram of association analysis, wherein the spots represent SNP sites;
FIG. 1B is a gene model of candidate gene MeMYB4 and the position of position SNP 2-6158256, where the vertical arrow indicates the GWAS signal and the asterisk indicates position SNP 2-6158256, which is significantly associated with root tuber endothelial color, located in the promoter region of MeMYB4 (i.e., 661bp upstream of the start codon);
FIG. 1C shows the cyanidin-3-O-glucoside content in cassava tubers at the SNP 2-6158256 locus of different genotypes (CC, TT and CT), the numbers in brackets indicate the number of samples for each genotype;
FIG. 1D shows the content of delphinidin-3-O-rutinoside in cassava tubers at the SNP 2-6158256 locus of different genotypes (CC, TT and CT), the numbers in brackets indicate the number of samples for each genotype;
FIG. 1E is a graph showing the percentage of the color of the root endothelium of cassava at the SNP 2-6158256 locus for different genotypes (CC, TT and CT), the numbers in brackets indicate the number of samples for each genotype.
FIG. 2 is a schematic diagram showing the typing of different genotypes (CC, TT and CT) of SNP loci according to the invention.
Detailed Description
The present invention is described in further detail below in conjunction with specific embodiments for understanding by those skilled in the art.
Example 1 based on genome-wide association analysis, SNP loci that are significantly associated with the color and anthocyanin content of the cassava root endothelium are mined
In the agricultural rural area, a leaf tissue of 299 parts of cassava germplasm is arbitrarily selected, genomic DNA is extracted by using a DNeasy Plant Mini kit (Qiagen, beijing), and then a pair-end sequencing library with an insert length of 500bp is constructed. Cassava genome re-sequencing was performed using the Illumina X-Ten platform, sequencing reads (reads) were aligned to the cassava SC205 reference genome using BWA mem v0.7.17 software, and sequencing and tag repeat processing was performed using Samtools v1.9 and Picard v1.94 software.
After removal of the linker sequence and low quality sequences, the SNPs were identified using GATK tool v3.5 software for sequences on single-ended and double-ended alignments. The initial genotype file containing SNP and Indel is established by adopting a HaplotyCaller module and is filtered, and the specific parameters are as follows: "QUAL <2.0 ] QD <2.0 ] MQ <40.0 ] FS >60.0 ] MQRankSum < -12.5 ] ReadPosRankSum < -8.0-clusterize 2-clusterindowSize 5" and "QD <2.0 ] FS >200.0 ] ReadPosRankSum < -20.0". The SNPs and indels identified were annotated using the snpiff v3.6c program. Filtering low quality SNPs with "major allele frequency >0.05 and deletion data <20%" ultimately produced 1,313,775 high quality SNPs for whole genome association analysis.
The anthocyanin content in the cassava tuberous root is measured by adopting a metabonomic method, and the anthocyanin content comprises the content of Cyanidin-3-O-glucoside (Cyanidin-3-O-glucoside) and Delphinidin-3-O-rutinoside (Delphinidin-3-O-rutinoside). About 80mg of ground cassava tubers were selected for each material, extracted overnight with 1.0mL of 70% aqueous methanol at 4℃and subsequently analyzed using an LC-ESI-MS/MS system (HPLC, shim-pack UFLC SHIMADZU CBM A system; MS, applied Biosystems 6500+Q TRAP). HPLC used a Waters ACQUITY UPLC HSS T C18 column (1.8 μm,2.1mm x 100 mm) with a solvent system of water (containing 0.04% acetic acid) and acetonitrile (containing 0.04% acetic acid) with the procedure: 0 min 95:5V/V,11 min 5:95V/V,12 min 5:95V/V,15 min 95:5V/V. The flow rate was 0.35mL/min and the injection volume was 2.0. Mu.L. ESI source usage parameters: the ion source is turbine spray; the source temperature was 500 ℃; the ion spraying voltage is (+) 5500V and (-) 4500V; ion source gas I (GSI), gas II (GSII), and curtain gas (curtaingas) were set at 55, 60, and 35psi, respectively; the collision gas (collision gas) is set to be medium. In the linear ion trap and triple quaternary rod (QQQ) modes, instrument tuning and mass calibration were performed using 10 and 100 μmol/L polyethylene glycol solutions, respectively. A series of specific MRM (multiple reaction monitoring) transitions were monitored based on the metabolites eluted during each period. The color of the inner skin of the cassava tubers is distinguished into white and red according to naked eyes.
And then carrying out whole genome association analysis by using the color of the cassava root endothelium as a phenotypic character and using a mixed linear model. The relative (K) matrix was constructed using SPAGEDI v1.3a software, setting the P value to 4.43e-07.
The result shows that: the 1 st chromosome 6.68-6.98Mb interval and the 2 nd chromosome 5.87-6.17Mb interval are significantly associated with the color of the cassava root endothelium (FIG. 1A).
Based on genome annotation, one gene, meMYB4, regulating anthocyanin synthesis was found in the 2 nd chromosome candidate gene interval. Furthermore, there was a SNP mutation site SNP 2-6158256 (FIG. 1B) at 661bp upstream of the MeMYB4 start codon, which was significantly correlated with anthocyanin metabolites (cyanidin-3-O-glucoside and delphinidin-3-O-rutinoside) and cassava root endothelia color.
The method comprises the following steps: compared with TT and CT genotypes, the content of cyanidin-3-O-glucoside and delphinidin-3-O-rutinoside in cassava tuberous root containing CC genotypes is obviously improved (figures 1C-D), and the proportion of red color of the tuberous root endothelium is obviously increased (figures 1E and P < 0.001). Through selecting the CC genotype of the SNP locus, the cassava material with high Yan Zhi (red endothelium) and high anthocyanin content can be rapidly selected, so that the selection efficiency is remarkably improved, and the breeding process is accelerated.
EXAMPLE 2SNP2_6158256 site SNP marker development
Based on a cassava SC205 reference genome, extracting 500bp sequences of the upstream and downstream of the SNP 2-6158256 locus respectively, adopting a Primer 5 to design a specific Primer pair rc1,
primer rc1_f:5'-TCTTACAATGGGATAAATTAGTGTTATTA-3' (SEQ ID No. 2),
primer rc1_r:5'-TTGGGTAGTTAGGTAGATGGGTTT-3' (SEQ ID No. 3);
the nucleotide sequence containing the SNP 2-6158256 locus is obtained through PCR amplification, namely the SNP marker related to the color of the inner skin of the cassava root and the anthocyanin content, and the nucleotide sequence is shown as SEQ ID No. 1.
TCTTACAATGGGATAAATTAGTGTTATTAATTAGGTTTCCTCTATATTATATATATTTCCTATAAAAAATAATTGGATTCAGATGTGTGGAAGCATAAATGCCAATTAGGAGCTTTCAGAAAATTTTAACATAGATGAAGAACTTCGATCTTTCCCGTCAACACACCAAGAAAAATAAATAAGCTATTGATTAAACTCATGAACTGCAATACTGTAGGAGCTGAATGTTCATATTTATTCTGAGATAAAAATTGTGAAATCY(C/T)CCACCACTTAATTCTTAATGTTAATTATAACTCA GTACTCTTCTGCTAAGTTGGCCTAATTATTTCTGAATTAATACT CCAATAAAGACCACACTTAACCTAATTTGAAGTTTGAAATTTA AAACCCATCTACCTAACTACCCAA。
Example 3
A kit for screening or detecting cassava material with high anthocyanin content and red root endothelia, comprising a specific primer pair rc1:
primer rc1_f:5'-TCTTACAATGGGATAAATTAGTGTTATTA-3' the number of the individual pieces of the plastic,
primer rc1_r:5'-TTGGGTAGTTAGGTAGATGGGTTT-3'.
The using method of the kit is as follows:
1) Extracting DNA of a cassava sample to be detected;
2) Carrying out PCR amplification by using a specific primer pair rc1 to obtain a PCR product; wherein, the liquid crystal display device comprises a liquid crystal display device,
the PCR reaction system is as follows: DNA template 2. Mu.l, PCR mix 25. Mu.l, primer rc1_F2mu.l, primer rc1_R2mu.l, d 2 H 2 O19. Mu.l, total 50. Mu.l;
the PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 53℃for 30sec, elongation at 72℃for 30sec,38 cycles; extending at 72 ℃ for 7min;
3) The PCR amplification products were detected by 1% agarose gel electrophoresis and then sequenced. And then checking a sequencing peak diagram, analyzing the genotype of the cassava sample to be tested, wherein,
when the 262bp position of the PCR product is T unimodal, the sample to be detected is TT genotype,
when the 262bp position of the PCR product is CT double peak, the sample to be detected is CT genotype,
when the 262bp position of the PCR product is C single peak, the sample to be detected is CC genotype (figure 2); the sample to be detected of the CC genotype is the cassava material with high anthocyanin content and red root tuber endothelium, and can be used for creating new cassava materials.
Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (8)
1. A SNP marker associated with the color and anthocyanin content of the inner skin of cassava tubers, characterized in that: the nucleotide sequence of the SNP marker is shown as SEQ ID No. 1; a SNP locus C/T is arranged at 262bp of the nucleotide sequence of the SNP marker; this site was designated as the chr2:6158256 site.
2. A specific primer set for obtaining the SNP marker according to claim 1, characterized in that: the primer pair rc1 is as follows:
primer rc1_f:5'-TCTTACAATGGGATAAATTAGTGTTATTA-3' the number of the individual pieces of the plastic,
primer rc1_r:5'-TTGGGTAGTTAGGTAGATGGGTTT-3'.
3. A method for obtaining the SNP marker according to claim 1, characterized in that: PCR amplification is carried out by taking cassava genome DNA as a template and the following primer pairs;
primer rc1_f:5'-TCTTACAATGGGATAAATTAGTGTTATTA-3' the number of the individual pieces of the plastic,
primer rc1_r:5'-TTGGGTAGTTAGGTAGATGGGTTT-3';
obtaining SNP marker, the nucleotide sequence of which is shown as SEQ ID No. 1.
4. A kit for screening or detecting cassava material with high anthocyanin content and red root endothelia, which is characterized in that: the kit contains the specific primer pair rc1 as defined in claim 2.
5. Use of the kit of claim 4 for screening or detecting cassava material with high anthocyanin content and red root endothelia.
6. A method of using the kit of claim 4, wherein: the method comprises the following steps:
1) Extracting DNA of a cassava sample to be detected;
2) Carrying out PCR amplification by using a specific primer pair rc1 to obtain a PCR product;
3) Detecting the PCR amplification product by using 1% agarose gel electrophoresis, and sequencing; and then checking a sequencing peak diagram, analyzing the genotype of the cassava sample to be tested, and screening to obtain the cassava material with high anthocyanin content and red root endothelia.
7. The method of use according to claim 6, wherein: in the step 2) of the above-mentioned process,
the PCR reaction system is as follows: DNA template 2. Mu.l, PCR mix 25. Mu.l, primer rc1_F2mu.l, primer rc1_R2mu.l, d 2 H 2 O19. Mu.l, total 50. Mu.l;
the PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 53℃for 30sec, elongation at 72℃for 30sec,38 cycles; extending at 72℃for 7min.
8. The method of use according to claim 6, wherein: in the step 3), the following results are obtained according to the sequencing comparison:
when the 262bp position of the PCR product is T unimodal, the sample to be detected is TT genotype,
when the 262bp position of the PCR product is CT double peak, the sample to be detected is CT genotype,
when the 262bp position of the PCR product is C single peak, the sample to be detected is CC genotype; the sample to be tested of the CC genotype is cassava material with high anthocyanin content and red root endothelia.
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| CN116949063B (en) * | 2023-09-12 | 2023-12-12 | 中国热带农业科学院三亚研究院 | Low-temperature response transcription factor and application thereof |
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