An Indel marker related to arsenic content in corn kernels and its application

Abstract

The present invention discloses an Indel marker related to arsenic content in corn kernels and its application, and relates to the technical field of plant genetic engineering. The InDel marker comprises an insertion sequence and a deletion sequence of a base as shown in SEQ ID NO.5, and the insertion or deletion position of the base is located at the +383rd position of the promoter of the ZmFADS1 gene, and the primer sequence for verifying the InDel marker is shown in SEQ ID NO.3‑4. The Indel marker provided by the present invention is significantly correlated with the arsenic content in corn kernels. If the InDel marker is an insertion type, the arsenic content in the corn kernels is low; if the InDel marker is a deletion type, the arsenic content in the corn kernels is high. The Indel marker can be used for molecular assisted breeding of corn and identification of high-quality planting resources, thereby accelerating the creation of low-arsenic enriched materials for corn kernels and the process of breeding new varieties.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to an Indel marker related to arsenic content in corn kernels and an application thereof. Background Art

[0002] With the rapid development of industry and modern agriculture, a large number of farmlands around the world are contaminated by heavy metals. Heavy metals in the soil will be absorbed by crops and accumulated in edible parts, threatening animal and human health through the food chain. Corn (Zea mays L.) is the world's largest food crop. The heavy metal arsenic content in corn kernels is directly related to its nutritional quality and food safety. Therefore, reducing the arsenic content in kernels is of great significance to ensuring future food safety. The exploration and use of molecular markers related to the arsenic content in kernels is of great practicality for screening low-arsenic germplasm in kernels and applying it to breeding.

[0003] Based on the research of the prior application (application number 2024112448830), we discovered the GRMZM2G381933 gene (named ZmFADS1 gene) in corn. The ZmFADS1 gene encodes the zeaxanthin adenine dinucleotide synthetase gene. Studies have found that the ZmFADS1 gene is overexpressed in corn to increase the ability of plant leaves to synthesize FAD, thereby increasing the ability of plant leaves to accumulate heavy metal arsenic, thereby reducing the arsenic content in corn kernels. We conducted further research based on the ZmFADS1 gene and discovered an Indel marker related to the arsenic content in corn kernels and its application. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the present invention aims to provide an Indel marker related to arsenic content in corn kernels and its application.

[0005] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0006] The present invention provides an Indel marker related to arsenic content in corn grains and an application thereof. The InDel marker comprises an insertion sequence and a deletion sequence of 186 bp bases as shown in SEQ ID NO.5, and the insertion or deletion position of the 186 bp bases is located at the +383rd position of the ZmFADS1 gene promoter. The ZmFADS1 gene promoter sequence is shown in SEQ ID NO.1 or 2, and the primer sequence for verifying the InDel marker is shown in SEQ ID NO.3-4.

[0007] An application of the Indel marker in identifying the arsenic content of corn kernels, wherein if the InDel marker is of insertion type, the corn variety has a low arsenic content in the corn kernels; and if the InDel marker is of deletion type, the corn variety has a high arsenic content in the corn kernels.

[0008] As a further optimization scheme of the present invention, if the InDel marker is an insertion type, the ZmFADS1 gene promoter activity and the ZmFADS1 gene expression level of the corn variety are high, so that the arsenic content of the corn grains is low; if the InDel marker is a deletion type, the ZmFADS1 gene promoter activity and the ZmFADS1 gene expression level of the corn variety are low, so that the arsenic content of the corn grains is high.

[0009] As a further optimization scheme of the present invention, the InDel marker sequence of the corn variety with the deletion type InDel marker is shown as SEQ ID NO.1, and the InDel marker sequence of the corn variety with the insertion type InDel marker is shown as SEQ ID NO.2.

[0010] A method for identifying the arsenic content of corn kernels using the InDel marker comprises the following steps:

[0011] S1, extracting the corn DNA to be tested;

[0012] S2, using a sequence containing the site where the InDel marker is located and its upstream and downstream bases as an amplification template, and performing PCR amplification using primers that verify the InDel marker to obtain an amplification product containing the site where the InDel marker is located;

[0013] S3, performing genotyping detection on the amplified product to obtain the InDel marker type of the corn variety to be tested;

[0014] If the InDel marker type of the corn to be tested is ZmFADS1-383-ins, the corn to be tested is a corn variety with low arsenic content in corn kernels;

[0015] If the InDel marker type of the corn to be tested is ZmFADS1-383-del, the corn to be tested is a corn variety with a high arsenic content in corn kernels.

[0016] The present invention provides the following beneficial effects:

[0017] The present application obtains the P1 haplotype (ZmFADS1-383-del) and P2 haplotype (ZmFADS1-383-ins) by analyzing the promoter sequence differences of the ZmFADS1 gene of corn inbred lines with different high / low arsenic contents in grains. The two haplotypes are further associated with the grain arsenic content and it is found that the arsenic content in the P2 haplotype corn grains is significantly lower than that in the P1 haplotype corn grains. InDel markers are developed based on these two haplotypes. The bands of the InDel markers are clearly distinguishable after electrophoresis detection. The band types of corn varieties with different high / low arsenic contents in grains are obviously different and easy to distinguish. The detection method is simple and can be used for molecular assisted breeding of corn and identification of high-quality planting resources, thereby accelerating the creation of low-arsenic enriched materials in corn grains and the process of breeding new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram of the screening and analysis results of candidate genes;

[0019] Figure 2 This is the expression analysis of genes after arsenic treatment;

[0020] Figure 3 It is the sequence analysis diagram and expression analysis diagram of ZmFADS1 gene of maize inbred line 78371A and maize inbred line ji434;

[0021] Figure 4 It is a comparison diagram of the promoter sequence differences of ZmFADS1 gene between maize inbred line 78371A and maize inbred line ji434;

[0022] Figure 5 This is the Motif analysis diagram of the 186 bp base inserted by the InDel in the promoter of the ZmFADS1 gene;

[0023] Figure 6 It is the LUC / REN value analysis chart and grain arsenic content analysis chart of corn inbred line 78371A and corn inbred line ji434;

[0024] Figure 7 This is an agarose gel electrophoresis pattern. DETAILED DESCRIPTION

[0025] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0026] 1. Materials

[0027] The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used are commercially available products unless otherwise specified.

[0028] 2. Methods

[0029] 2.1 Screening of candidate genes

[0030] The national minimum safety standard for arsenic content in corn kernels is 0.2 mg / Kg. This application tested the arsenic content of 256 different corn inbred line kernel materials and found that the arsenic content in the kernels was 0.007-0.28 mg / Kg (e.g. Figure 1 A genome-wide association analysis of arsenic content in grains was performed on 256 different maize inbred lines, and a significant polymorphic site (SNP) was found at position 4617696 of chromosome 8. Ten SNPs within 100 kb of this region were associated with arsenic content in grains (as shown in Figure 1 b, c);

[0031] Wild-type maize B104 seedlings 10 days after sowing were treated with different concentrations of arsenic (0mM, 0.1mM, 0.5mM, 1.0mM, 5.0mM, and 10mM AsIII) for three days. RNA was then extracted from the wild-type maize B104 seedlings after arsenic treatment to obtain cDNA, and QRT-PCR (real-time fluorescence quantitative PCR) was used to identify 12 candidate genes in this interval (such as Figure 1 d) and found that the expression of gene GRMZM2G381933 (named as ZmFADS1 gene) was significantly induced by arsenic. With the increase of arsenic concentration, its expression level was significantly upregulated (as shown in Figure 2 a);

[0032] Ten days after sowing, wild-type maize inbred line B104 was treated with 1.0 mM AsIII at 0 h, 6 h, 15 h, 18 h and 24 h. It was found that the gene GRMZM2G381933 (named as ZmFADS1 gene) was significantly induced by arsenic, and the expression level of the gene GRMZM2G381933 (named as ZmFADS1 gene) was the highest at 6 h of treatment (e.g. Figure 2 b).

[0033] 2.2 Haplotype analysis of candidate genes

[0034] 103 maize inbred lines were randomly selected from the GWAS population, and the ZmFADS1 gene was resequenced. The promoter plus gene length is about 6.5 kb. A genome-wide association analysis was performed on the full length of the gene and the arsenic content in the grain. It was found that an Indel (a 186 bp base insertion or deletion at the 383 bp position of the promoter of the ZmFADS1 gene) was significantly correlated with the arsenic content in the maize grain (P = 1.06 x 10-9; P = 0.01). Figure 3 The SNPs and Indels in this region have a strong linkage disequilibrium relationship (R2>0.8; as shown in a). Figure 3 a). This indicates that the arsenic content in the grain is significantly associated with Indel. According to the analysis results, the 103 maize inbred lines were divided into two haplotypes, the haplotype P1 without Indel (n=45) and the haplotype P2 with Indel (n=58). ZmFADS1-78371A is a representative of the P1 haplotype, and the arsenic content in the grains of the inbred line 78371A is relatively high (0.27 mg / kg), exceeding the national standard of 0.20 mg / kg; ZmFADS1-ji434 is a representative of the P2 haplotype, and the arsenic content in the grains of the inbred line ji434 (JIAO, Y., ZHAO, H., REN, L., SONG, W., ZENG, B., GUO, J., WANG, B., LIU, Z., CHEN, J., LI, W., ZHANG, M., XIE, S. & LAI, J. 2012. Genome-wide genetic changes during modern breeding of maize. Nat Genet, 44, 812-5.) is low (0.07 mg / kg).

[0035] Based on the promoter and full-length sequence analysis of the ZmFADS1 gene, the P1 and P2 haplotypes were further analyzed for association with the grain arsenic content (e.g. Figure 3b, **P<0.01), the arsenic content in P2 haplotype corn (186bp base insertion at the +383bp position of the promoter of the ZmFADS1 gene) was significantly lower than that in P1 haplotype corn (186bp base deletion at the +383bp position of the promoter of the ZmFADS1 gene); the present application also analyzed the transcriptome of the published corn NAM inbred line population (MCMULLEN, MD, KRESOVICH, S., VILLEDA, HS, BRADBURY, P. ,LI,H.,SUN,Q.,FLINT-GARCIA,S.,THORNSBERRY,J.,ACHARYA,C.,BOTTOMS,C.,BROWN,P.,BROWNE,C.,ELLER, M.,GUILL,K.,HARJES,C.,KROON,D.,LEPAK,N.,MITCHELL,SE,PETERSON,B.,PRESSOIR,G.,ROMERO,S.,OROPEZA ROSAS, M., SALVO, S., YATES, H., HANSON, M., JONES, E., SMITH, S., GLAUBITZ, JC, GOODMAN, M., WARE, D., HOLLAND, JB & BUCKLER, ES 2009. Genetic properties of the maize nested association mapping population. Science, 325, 737-40.), and found that the expression of the ZmFADS1 gene in the P2 haplotype was higher than that in the P1 haplotype (e.g. Figure 3 c, ***P<0.001).

[0036] Sequence analysis of the ZmFADS1 gene promoter region of the kernel materials of the maize inbred line 78371A with the highest kernel arsenic content and the maize inbred line ji434 with the lowest kernel arsenic content was performed. Figure 4 The promoter sequence corresponding to the P1 haplotype is shown in SEQ ID NO.1, and the promoter sequence corresponding to the P2 haplotype is shown in SEQ ID NO.2.

[0037] Further sequence analysis revealed that the 186 bp base contained two GT1-motifs, three Sp1-motifs, one GC-motif, one ABRE-motif, one chs-Unit 1m1-motif and one as-1-motif (e.g. Figure 5These Motifs are mainly related to the response to light signals; it is speculated that this region may be regulated by related transcription factors, thereby increasing its promoter activity and further increasing the expression level of the ZmFADS1 gene.

[0038] In the present application, the promoters of ZmFADS1-78371A (sequence as shown in SEQ ID NO.1) and ZmFADS1-ji434 (sequence as shown in SEQ ID NO.2) were constructed in front of the LUC gene of the pGreenII0800 vector, and the constructed plasmids were transferred into corn protoplasts to measure the LUC / REN value. The analysis found that the LUC / REN value of the promoter of ZmFADS1-ji434 was significantly higher than that of ZmFADS1-78371A (sequence as shown in SEQ ID NO. Figure 6 a), indicating that the promoter activity of ZmFADS1-ji434 was significantly higher than that of ZmFADS1-78371A, proving that the 186 bp Indel insertion increased the promoter activity of the ZmFADS1 gene, thereby increasing the expression level of the ZmFADS1 gene and significantly reducing the arsenic content in corn kernels (as shown in Figure 6 b).

[0039] 2.3 Development of molecular markers based on candidate genes

[0040] Based on the conclusion obtained in step 2.2 above (the insertion / deletion of 186 bp base at the +383 bp position of the promoter of the ZmFADS1 gene is extremely significantly correlated with the arsenic content in corn kernels), specific primers were designed for the sequence difference of the InDel (ZmFADS1-383-ins / del) to develop an InDel marker. The specific primer sequences are shown in SEQ ID NO.3-4:

[0041] SEQ ID NO.3: ZmFADS1-F:5'-GGAGCATCATACGAGCCA-3';

[0042] SEQ ID NO.4: ZmFADS1-R:5'-CGCACCCTGAATCTCGAAC-3'.

[0043] DNA of two haploid maize materials, namely maize inbred line 78371A with the highest arsenic content in kernels and maize inbred line ji434 with the lowest arsenic content, was extracted and used as templates for PCR amplification. PCR amplification was performed using designed specific primers and high-fidelity enzyme KOD FX neo to obtain different gene fragments. The results are as follows: Figure 7As shown, the length of the P2 haplotype band is significantly greater than that of the P1 haplotype band, which is consistent with the sequencing analysis results that the P1 haplotype (ZmFADS1-383-del) is 1468 bp and the P2 haplotype (ZmFADS1-383-ins) is 1658 bp.

[0044] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An application of an InDel marker in identifying the arsenic content in corn kernels, characterized in that: The nucleotide sequence of the InDel marker is shown in SEQ ID NO.1-2; When the nucleotide sequence of the InDel marker is as shown in SEQ ID NO.1, the arsenic content of the corn kernels of the tested corn variety is high; When the nucleotide sequence of the InDel marker is as shown in SEQ ID NO. 2, the arsenic content of corn kernels of the tested corn variety is low.

2. A method for identifying arsenic content in corn kernels using InDel markers, characterized in that: The following steps are involved: S1, extracting the corn DNA to be tested; S2, using the sequence containing the site where the InDel marker is located and the sequence of the upstream and downstream bases as the amplification template, designing primers for the InDel marker, and performing PCR amplification to obtain an amplification product containing the site where the InDel marker is located; The nucleotide sequence of the InDel marker is shown in SEQ ID NO.1-2; The nucleotide sequence of the InDel-labeled primer is shown in SEQ ID NO.3-4: SEQ ID NO.3: Upstream primer: 5'-GGAGCATCATACGAGCCA-3'; SEQ ID NO.4: Downstream primer: 5'-CGCACCCTGAATCTCGAAC-3'; S3, performing genotyping detection on the amplified product to obtain the InDel marker type of the corn variety to be tested; If the type of the InDel marker of the corn to be tested is as shown in SEQ ID NO.1, the arsenic content of the corn kernels of the corn variety to be tested is high; If the InDel marker type of the corn to be tested is as shown in SEQ ID NO.2, the arsenic content of corn kernels of the corn variety to be tested is low.

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