A SNP site affecting cassava drought resistance and its application

Abstract

The present invention provides a SNP site, which is located at the 296th position of the coding region of the MeGRXC4 gene, and the base is G or C. The sequence of the coding region of the MeGRXC4 gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2. The present invention identifies for the first time from a big data level that the SNP located at the 296th position of the MeGRXC4 nucleotide sequence is closely related to the drought resistance of cassava, is conducive to detection, is accurate and reliable, and is easy to operate. The drought resistance of cassava is identified by using the SNP site, which has high accuracy and is not affected by the growth period of cassava. It can be selected in the seedling stage, which significantly improves the breeding efficiency. In addition, when the base of the SNP site is G, MeGRXC4 can significantly improve the drought resistance of cassava. The present invention is of great significance for revealing the relationship between allele selection and the formation of drought resistance traits in the process of cultivating cassava domestication, and provides gene resources and scientific basis for breeding new drought-resistant and high-yield cassava varieties using key genes.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to a SNP site affecting cassava drought resistance and an application thereof. Background Art

[0002] Cassava (scientific name: Manihot esculenta Crantz) is rich in starch and is one of the raw materials for industrial starch. Although cassava can be cultivated in a rough manner and yields can be obtained, it is crucial to cultivate varieties with high resistance in order to achieve high-yield, high-quality and efficient cultivation results. Although cassava is drought-resistant and avoids waterlogging, long-term drought, especially water shortage during the cassava tuber formation period 60-70 days after planting, will inevitably affect its absorption of nutrients, thereby hindering its reproduction and production and affecting yield. Therefore, improving cassava drought resistance is crucial for the cassava planting industry. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a SNP site affecting cassava drought resistance and its application.

[0004] The first aspect of the present invention is to provide a SNP site, wherein the SNP site is located at position 296 in the coding region of the MeGRXC4 gene, the base is G or C, and the sequence of the coding region of the MeGRXC4 gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0005] The inventors found that the drought resistance of cassava with the GG genotype at the SNP site is significantly better than that of cassava with the CC genotype and cassava with the GC genotype.

[0006] The second aspect of the present invention is to provide a method for detecting drought resistance of cassava in the seedling stage, extracting genomic DNA of the cassava to be tested, detecting the SNP sites described in the first aspect of the present invention, determining the genotype of the cassava to be tested, and thus determining the drought resistance of the cassava to be tested.

[0007] When the genotype of the SNP site is GG, it is identified that the cassava has good drought resistance, and when the genotype of the SNP site is CC genotype or GC genotype, the cassava has poor drought resistance.

[0008] The third aspect of the present invention is to provide a cassava breeding method, which comprises extracting genomic DNA of the cassava to be tested, detecting the SNP sites described in the first aspect of the present invention, determining the genotype of the cassava to be tested, thereby determining the drought resistance of the cassava to be tested, and breeding the cassava.

[0009] The fourth aspect of the present invention is to provide the use of the SNP site described in the first aspect of the present invention, or the method described in the second aspect of the present invention, or the breeding method described in the third aspect of the present invention in cassava breeding.

[0010] The fifth aspect of the present invention is to provide the use of the MeGRXC4 gene, or the protein encoded by the MeGRXC4 gene, or a recombinant vector or host bacteria or expression cassette containing the MeGRXC4 gene coding region in improving the drought resistance of cassava, wherein the coding region sequence of the MeGRXC4 gene is shown in SEQ ID NO: 2, and the base of the SNP site described in the first aspect of the present invention is G.

[0011] The original vector of the recombinant vector can be a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc. The present invention is not limited to this. In a specific embodiment of the present invention, the original vector of the recombinant vector is a PMD18-T vector or a pCAMBIA1300 vector.

[0012] The sixth aspect of the present invention is to provide a gene fragment for improving the drought resistance of cassava. The coding region sequence of the gene fragment is shown in SEQ ID NO: 2. The base of the SNP site in the first aspect of the present invention is G.

[0013] The eighth aspect of the present invention is to provide a primer pair for amplifying the gene fragment described in the sixth aspect of the present invention, wherein the primer pair is MeGRXC4-G99-F: GGAGTCGAC ATG GAG GAG CAG AAG CTG ATC and MeGRXC4-G99-R: CAAGGATCC CTA AAT CCA TAT AGC TCC TGC ATT; or the primer pair is MeGRXC4-G99-Forward: CAACCATGG ATG GAG GAG CAG AAG CTG ATC and MeGRXC4-G99-Reverse: GGAGTCGAC CTA AAT CCA TAT AGC TCC TGC.

[0014] The present invention identifies for the first time from a big data level that the SNP at the 296th position of the MeGRXC4 nucleotide sequence is closely associated with the drought resistance of cassava, is conducive to detection, is accurate and reliable, and is easy to operate. The SNP site provided by the present invention is used to identify the drought resistance of cassava with high accuracy. Moreover, the SNP site and the detection method provided by the present invention are not affected by the growth period of cassava, and can be selected in the seedling stage, which greatly reduces the breeding workload, significantly shortens the breeding selection cycle, and improves the breeding efficiency. In addition, when the base of the SNP site is G, MeGRXC4 can significantly improve the drought resistance of cassava. The present invention is of great significance for revealing the relationship between allele selection and the formation of drought resistance traits in the process of cultivating cassava domestication, and provides gene resources and scientific basis for cultivating new drought-resistant and high-yield cassava varieties using key genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Among 100 cultivated cassava germplasm populations, MeGRXC4 Association analysis between relevant SNPs and drought resistance indicators. (a) MeGRXC4 Association plots of promoter and coding region SNPs with drought resistance indicators. The English letters represent the abbreviations of drought resistance indicators. -L represents leaves, -R represents roots, and -2014 and -2015 represent the data collection years of 2014 and 2015, respectively (drought resistance indicator data have been published, see reference Wang et al. , PlosOne, 2017). (b) Analysis of the G99A mutation site in the MeGRXC4 coding region. The gray area is the gene coding region, ATG is the start codon, and TAG is the stop codon; the codon corresponding to G99 is GG 296 T, encoding glycine (Gly); the codon corresponding to A99 is GC 296 T, encoding alanine (Ala); 5'UTR: 5' untranslated region; 3'UTR: 3' untranslated region. (c) The distribution of SNPs corresponding to the G99A site in the genomes of 100 cultivated cassava germplasms.

[0016] Figure 2 The distribution of the SNP corresponding to the MeGRXC4 protein G99A mutation site in the genome of 244 cassava germplasms, of which 135 germplasms were homozygous for G99 / G99, 21 germplasms were homozygous for A99 / A99, and 88 germplasms were heterozygous for G99 / A99.

[0017] Figure 3 Phenotypic analysis of drought resistance of transgenic cassava. D0: the day of water cessation; D10: 10 days of water cessation; the numbers on the right side of the survival rate are the number of potted seedlings.

[0018] Figure 4 MeGRXC4 protein mutation site G99A MeGRXC4 Functional effects of regulating reporter gene expression in yeast. DETAILED DESCRIPTION

[0019] The present invention will be further described below with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be obtained commercially.

[0020] Example 1: Acquisition of SNP loci associated with cassava drought resistance

[0021] The seed stems of 100 cassava germplasms were cut into stem segments with relatively uniform length, cut into pots filled with an equal amount of soil: sand 2:1 mixed substrate, and placed in a rainproof shed for cultivation. At 100 days after cutting, 12 seedlings with relatively consistent growth potential were selected for drought treatment and normal watering control group treatment. The drought treatment method was to stop watering for 12 days in the drought treatment group, and the control group was watered regularly and quantitatively every day. After 12 days of stopping watering, 3 plants with relatively consistent growth potential and drought performance were selected from each variety / strain as the objects of investigation of the treatment group / control group. The aboveground fresh weight, tuber fresh weight, tuber number, tuber dry weight and tuber dry matter rate of the treatment group and the control group were investigated respectively, and 1-2 leaves were taken from the upper, middle and lower parts of the three plants, cut into pieces, mixed, and leaf samples were taken. The roots of the three plants were first mixed, cut into pieces, sampled, placed in liquid nitrogen for quick freezing, and stored in a -80℃ refrigerator for physiological index detection. Physiological index detection mainly includes proline content, soluble reducing sugar content, malondialdehyde content, SOD activity, POD activity and CAT activity. The detection of 6 physiological indexes was completed at Suzhou Keming Biotechnology Co., Ltd. The proline content was detected by ninhydrin colorimetry, the soluble reducing sugar content was detected by anthrone colorimetry, the malondialdehyde content was detected by TBA method, the SOD activity was detected by NBT method, the POD activity was detected by guaiacol method, and the CAT activity was detected by H2O2 ultraviolet absorption method. For specific methods, please refer to the instructions for use of the corresponding kits of Suzhou Keming Company.

[0022] The drought resistance indexes were: fresh weight of aboveground part, fresh weight of tubers, number of tubers, dry weight of tubers, dry matter rate of tubers, proline content, soluble reducing sugar content, malondialdehyde content, SOD activity, POD activity and CAT activity. The drought resistance indexes of the same batch of cassava germplasm were tested for two years in 2014-2015 (repeated twice). SPSS18.0 software (http: / / www-01.ibm.com / software / analytics / spss / ) was used to perform descriptive statistical analysis on the phenotypic values, the difference significance analysis of the overall mean values ​​between the drought treatment group and the control group, and the Pearson correlation analysis of the phenotypic drought resistance coefficient of the phenotypic values. The descriptive statistical analysis included the maximum value, minimum value, mean value, standard deviation and coefficient of variation. The mean values ​​were compared using the one-way analysis of variance (ANOVA) method to output the F value and P value.

[0023] The MeGRXC3 gene region (including 2000bp upstream of ATG, gene region and 3'-UTR region) was resequenced using the multiplex PCR method (Chen et al. 2016). Multiplex PCR primers were designed to amplify the target region sequence from genomic DNA. The multiplex PCR reaction was optimized to ensure uniform coverage of the target region. The target region was amplified, sequencing adapters were added, and different samples were distinguished by different indexes. The target region was deeply sequenced using the high-throughput sequencing platform Illumina X-10 sequencing platform. The sequencing multiples were 30×~250× and the average sequencing depth was 225×. Read sequences were imported into FASTX-Toolkit (http: / / hannonlab.cshl.edu / fastx_toolkit / ) for quality control, and the amplicon sequences were aligned to the cassava genome Version 6.1 (https: / / phytozome.jgi.doe.gov / pz / portal.html#!info?alias=Org_Mesculenta, Bredeson et al. 2016) using BWA software (V0.7.12). SNP calling and low-quality SNP filtering were performed using GATK software (V3.8-0, https: / / software.broadinstitute.org / gatk / ). Multiplex PCR targeted resequencing was performed at Shanghai Biowing Applied Biotechnology Co. LTD. The SNP information was imported into snpEff software (http: / / snpeff.sourceforge.net / SnpEff_manual.html), and the obtained SNPs were functionally annotated using the cassava genome Version 6.1 sequence and annotation information.

[0024] Cassava drought resistance index was used as phenotype, EST-SSR marker genotypes of 100 cassava cultivars distributed in the cassava genome (Wang et al. 2017) were used to evaluate population structure and family effect as covariance, and SNP of MeGRXC4 was used as genotype, imported into TASSEL2.1 software (Yu et al. 2006), low-frequency SNP sites were eliminated under the condition of MAF>0.05, and candidate gene association analysis was performed using Q+K+MLM analysis model. The P value was obtained using the method of Bajamii et al. for false discovery rate control (FDR, Benjamini and Hochberg 1995). When P_FDR<0.05, it indicated that the SNP was significantly associated with the phenotype.

[0025] The results showed that in 100 cultivated cassava germplasm populations, the target interval resequencing results were associated with drought resistance indicators. MeGRXC4 There are multiple SNP sites in the promoter region that are significantly correlated with drought resistance indicators. Among them, a non-synonymous SNP (296G-C) appears in the codon corresponding to the 99th amino acid residue of the MeGRXC4 encoded protein ( Figure 1 ).

[0026] Example 2: Cassava drought resistance and target SNP site genotype analysis

[0027] Subsequently, based on the genome resequencing data of 244 natural cassava populations including wild species provided by the cooperative team, the SNP corresponding to the G99A variant site of the MeGRXC4 protein was analyzed. Figure 2 ). 244 cassava samples were subjected to drought treatment to detect drought resistance of the samples, and the detection method and standard were the same as those in Example 1. The results are shown in Table 1. Among the 244 cassava samples, 135 were homozygous for G99 / G99, all of which were drought-resistant samples, while 21 were homozygous for A99 / A99, and 88 were heterozygous for G99 / A99, all of which were not drought-resistant samples.

[0028] Table 1 Cassava drought resistance and target SNP locus genotype analysis

[0029] genotype Number of drought-resistant samples Number of samples not resistant to drought total G99 / G99 homozygous (G296 / G296) 135 0 135 A99 / A99 homozygous (C296 / C296) 0 21 21 G99 / A99 heterozygous (G296 / C296) 0 88 88

[0030] Example 3: Cloning, transformation and sequence analysis of MeGRXC4-G99 gene and MeGRXC4-A99 gene

[0031] RNA was extracted from mature leaves of cassava cultivar cv.60444 after drought treatment, and cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit, with DNase I (Thermo Scientific, USA). Using this cDNA as a template, PCR amplification of the MeGRXC4-A99 gene was performed using the primer pair MeGRXC4-A99-F: GGAGTCGACATG GAG GAG CAG AAG CTG ATC and MeGRXC4-A99-R: CAAGGATCC CTA AAT CCA TAT ACC TCCTGC ATT. PCR amplification of the MeGRXC4-G99 gene was performed using the primer pair MeGRXC4-G99-F: GGAGTCGAC ATG GAGGAG CAG AAG CTG ATC and MeGRXC4-G99-R: CAAGGATCC CTA AAT CCA TAT AGC TCC TGC ATT.

[0032] PCR reaction system: PrimeSTAR Max Premis (2´) premix (25 mL), forward primer (1 mL), reverse primer (1 mL), cDNA template (0.5 mL), deionized water (make up to 50 mL). PCR amplification program: 98°C denaturation for 10 s, 55°C annealing for 5 s, 72°C extension for 5 s, 35 cycles.

[0033] The amplified product was recovered, and the cloned DNA fragment was connected to PMD18-T (Beijing TaKaRa Company) for sequencing to obtain MeGRXC4-A99 gene and MeGRXC4-G99 gene. The coding region sequence of MeGRXC4-A99 gene is shown in SEQ ID NO: 1, and the SNP site corresponding to the G99A mutation site of MeGRXC4 protein is located at the 296 bp site from the 5' end of the sequence shown in SEQ ID NO: 1, and the base is C. The coding region sequence of MeGRXC4-G99 gene is shown in SEQ ID NO: 1, and the SNP site corresponding to the G99A mutation site of MeGRXC4 protein is located at the 296 bp site from the 5' end of the sequence shown in SEQ ID NO: 2, and the base is G.

[0034] Example 4: Analysis of drought resistance of transgenic plants

[0035] The MeGRXC4-G99 gene fragment and the MeGRXC4-A99 gene fragment cloned in Example 3 were respectively Sal I / BamThe recombinant plasmids 35S:MeGRXC4-G99-GFP and 35S:MeGRXC4-A99-GFP were obtained by double digestion with HI and inserted into the pCAMBIA1300 vector carrying GFP. The recombinant plasmids 35S:MeGRXC4-G99-GFP and 35S:MeGRXC4-A99-GFP were transformed into Agrobacterium tumefaciens LB4404, respectively.

[0036] Cassava embryonic callus suspension cell genetic transformation system mediated by Agrobacterium (LBA4404) using cultivated cassava cv.60444 as the recipient material. Specific steps:

[0037] 1) Collect Agrobacterium: Take the expanded Agrobacterium out of the shaker, measure OD600, collect the bacteria at 4℃, 6000 rpm for 10 min, and discard the supernatant; resuspend the bacteria in an appropriate amount of MS liquid (pH 5.3) MS medium to make the final concentration OD600 1.0;

[0038] 2) Place the callus in a MS culture bottle and shake culture for 45 minutes;

[0039] 3) The infected callus was transferred to a 100-mesh filter membrane. After air drying, the filter membrane was transferred to +AS (100 μM) solid culture medium and cultured in the dark at 23°C for 72 hours.

[0040] 4) Wash the co-cultured callus with MS liquid medium (pH 5.8), ( +Carb 500μg / ml);

[0041] 5) The infected suspension cells are plated on MSN medium containing Carb and Hyg to induce the production of resistant somatic embryos;

[0042] 6) Pick out the resistant somatic embryos and place them on CMM medium containing 5 μg / ml Hyg until resistant buds grow;

[0043] 7) The resistant shoots were cut off and transferred to CBM (Hyg 10 μg / ml) to form plants and take roots.

[0044] The single-copy transgenic plants that were positive after hygromycin resistance screening, PCR identification, Southern blot identification, RT-qPCR identification, and Western blot identification were used for phenotypic analysis.

[0045] The sieved peat soil and Hainan red soil were sterilized at 121℃ for 20 min to remove insect eggs and pathogens; black soil, vermiculite, red soil and perlite were mixed in a ratio of 10:10:5:3, and Bichongling, Avidamanling, Avermectin, rooting powder, HB101 particles and biochar powder were added during the mixing process.

[0046] Transplant the prepared cassava seedlings into the nutrient pots, fill them with the prepared nutrient soil, and make sure to compact it slightly to allow the roots to fully contact the soil; water until the soil is saturated with water; cover with a transparent lid (to prevent the leaves from losing too much water), and cultivate under weak light until the roots resume growth, then transplant to a larger pot.

[0047] 90 days after transplanting, we selected transgenic and wild-type cassava plants with similar growth and soil relative water content and stopped watering them for drought treatment. After 10 days of drought treatment, we calculated the survival rate. Figure 3 As shown, the survival rate of transgenic cassava with MeGRXC4-G99 gene reached 100%, while the survival rate of transgenic cassava with MeGRXC4-A99 gene was comparable to that of the wild type, at about 30%, indicating that the MeGRXC4-G99 gene can significantly improve the drought resistance of cassava.

[0048] Example 5: Yeast Experiment

[0049] Using the cDNA obtained in Example 3 as a template, PCR amplification of the MeGRXC4-G99 gene was performed using the primer pair MeGRXC4-G99-Forward: CAACCATGG ATGGAG GAG CAG AAG CTG ATC and MeGRXC4-G99-Reverse: GGAGTCGAC CTA AAT CCA TAT AGCTCC TGC, and PCR amplification of the MeGRXC4-A99 gene was performed using MeGRXC4-A99-Forward: CAACCATGG ATG GAGGAG CAG AAG CTG ATC and MeGRXC4-A99-Reverse: GGAGTCGAC CTA AAT CCA TAT ACC TCCTGC. The amplification reaction system and amplification procedure were referred to in Example 3.

[0050] The amplified product was recovered, and the obtained correct sequence was ligated to the pGBKT7 vector using NcoI / SalI double restriction enzymes, and yeast strain Y187 was transformed. The yeast transformation procedure was referred to the Clontech Laboratories Yeast Transformation System 2 User Manual. The transformed yeast strain was identified by PCR, and the positive clones were used for X-α-gal colorimetric reaction. 25ul of X-α-gal was applied to SD / -Trp plates, and the plates were dried. The positive clones were transferred to the plates for culture. After 2-3 days, the color development of the plates was observed and photographed. X-α-gal was used for colorimetric reaction. β -Gal activity analysis was performed and calculated according to the instructions of Clontech. Figure 4 As shown, MeGRXC4-G99 In yeast, the reporter gene expression cannot be activated (no blue spots), while MeGRXC4-A99 The expression of the reporter gene can be activated (obvious blue spots).

[0051] 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 modification and substitution of the practical is also within the scope of the present invention. Therefore, the equalization transformation and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for detecting drought resistance of cassava at the seedling stage, characterized in that: The genomic DNA of the cassava to be tested is extracted, and the SNP site is detected to determine the genotype of the cassava to be tested, thereby determining the drought resistance of the cassava to be tested, wherein the SNP site is located at the 296th position of the MeGRXC4 gene coding region, the base is G or C, the sequence of the MeGRXC4 gene coding region is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and when the genotype of the SNP site is CC genotype or GC genotype, the cassava drought resistance is poor.

2. A cassava breeding method, characterized in that: The genomic DNA of the cassava to be tested is extracted, and the SNP site is detected to determine the genotype of the cassava to be tested, thereby determining the drought resistance of the cassava to be tested, and breeding the cassava. The SNP site is located at the 296th position of the MeGRXC4 gene coding region, and the base is G or C. The sequence of the MeGRXC4 gene coding region is shown in SEQ ID NO: 1 or SEQ ID NO:

2. The drought resistance of the GG genotype cassava at the SNP site is better than that of the CC genotype and the GC genotype.

3. Use of a detection reagent for detecting a SNP site or the method according to claim 2 in cassava breeding, wherein the SNP site is located at position 296 of the MeGRXC4 gene coding region, the base is G or C, the sequence of the MeGRXC4 gene coding region is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the GG genotype of cassava at the SNP site has better drought resistance than the CC genotype and the GC genotype.

4. Use of a recombinant vector or host bacteria or expression cassette containing the MeGRXC4 gene in improving the drought resistance of cassava, wherein the coding region sequence of the MeGRXC4 gene is shown in SEQ ID NO:

2.

5. The use according to claim 4, characterized in that: The original vector of the recombinant vector is a PMD18-T vector or a pCAMBIA1300 vector.

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