SNP molecular marker combination and chip related to fresh corn nutritional element traits and application thereof

Abstract

The present application belongs to the technical field of gene chip, and particularly relates to a SNP molecular marker combination and chip related to fresh corn nutritional element traits and application thereof. Through whole genome association analysis, 257 SNP sites significantly associated with fresh corn nutritional elements (K, Mg, Ca, Fe, Zn, Mn, Cu and Ni) are screened, 20,000 SNPs uniformly distributed on the genome are selected in combination with SNPs identified by resequencing data, 21,768 SNP molecular marker combinations shown in Table 2 are obtained, and are tightly linked to fresh corn nutritional element content. Based on the above-mentioned SNP molecular marker combination, a probe is designed, a whole genome chip is constructed, and fresh corn genetic diversity and variety analysis can be carried out. Only by using the chip to identify the genotype of the fresh corn inbred line, the nutritional element content of the fresh corn inbred line can be predicted, and the application has the advantages of clear selection target, early detection and environmental influence.

Description

Technical Field

[0001] This invention belongs to the field of gene chip technology, specifically relating to a combination of SNP molecular markers and a chip related to the nutritional traits of fresh corn and their applications. Background Technology

[0002] Zinc (Zn) and iron (Fe) are essential / beneficial micronutrients for animals, plants, and humans. Low intake of zinc and iron, leading to malnutrition, is a significant global problem, especially in rural areas where grains are the staple food. Sweet corn refers to a special type of corn grown for its edible ears during the milk stage, mainly including sweet corn, waxy corn, and sweet-waxy corn. With the improvement of people's living standards, the market has placed higher demands on the quality of sweet corn. Currently, sweet corn breeding mainly relies on traditional phenotypic selection methods, which have limitations such as long breeding cycles, low efficiency, and difficulty in achieving multi-gene aggregation, making it difficult to meet current breeding needs.

[0003] Genome-wide selection breeding technology is one of the core technologies in the era of intelligent breeding for sweet maize. Its core idea is to estimate the breeding potential of individuals using molecular markers covering the entire genome, and then select individuals based on these predicted breeding potentials. This technology offers advantages such as shortening the breeding cycle, improving breeding efficiency, and achieving multi-gene aggregation, and is currently widely used in animal breeding, including dairy cows and pigs. However, the high cost of genotyping is a major factor limiting its widespread application in sweet maize breeding. Although several breeding chips have been developed in the maize field, the genetic basis of sweet maize differs significantly from that of ordinary grain maize because the breeding traits of sweet maize focus on taste and nutritional quality. Using breeding chips developed based on grain maize for genotyping of sweet maize often results in low locus detection diversity, limiting its applicability. Summary of the Invention

[0004] The purpose of this invention is to provide a combination of SNP molecular markers related to the nutritional traits of fresh maize, and to develop a breeding chip related to the nutritional traits of fresh maize kernels, which can predict the nutritional content of fresh maize with high accuracy. It can be used for whole-genome selection breeding of fresh maize and has the advantages of low cost, clear selection target, early detection and no environmental influence.

[0005] This invention provides a combination of SNP molecular markers related to the nutritional traits of fresh maize. The positions of the SNP molecular marker combination on the maize reference genome B73 RefGen_v4 version are shown in Table 2 of the specification.

[0006] This invention provides a probe for detecting the SNP molecular marker combinations described in the above-mentioned technical solutions.

[0007] Preferably, the probe is synthesized based on the location of the SNP site and the 200 bp sequence flanking it, with a GC content between 40% and 60%, a TM value ≥ 65°C, a sequence complexity LCC value ≥ 0.9, a number of identical base repeats ≤ 6, and the number of homologous regions of the probe sequence in the whole genome ≤ 3.

[0008] This invention provides a chip related to the nutritional characteristics of fresh corn, the chip being used to detect the molecular marker combinations described in the above technical solution.

[0009] Preferably, the chip is a liquid phase chip.

[0010] Preferably, the chip includes the probe described in the above technical solution.

[0011] This invention provides a whole genome sequencing kit for fresh corn, comprising the chip described in the above technical solution.

[0012] This invention provides the application of reagents for detecting the SNP molecular marker combinations described in the above-mentioned technical solutions, or probes described in the above-mentioned technical solutions, or chips described in the above-mentioned technical solutions, or fresh maize whole genome detection kits described in the above-mentioned technical solutions, in one or more of the following: (1) Analysis of target traits of fresh corn; the target traits include nutritional elements; (2) Breeding and / or assisted breeding of high-quality nutrient-rich fresh corn; (3) Analysis of genetic diversity in fresh maize; (4) Analysis of fresh corn varieties.

[0013] Preferably, the target trait analysis of fresh corn includes predicting the nutrient content of fresh corn.

[0014] Preferably, the nutrient elements include one or more of potassium, magnesium, calcium, iron, zinc, manganese, copper and nickel.

[0015] Beneficial effects: This invention, through genome-wide association study (GWAS) analysis, screened 257 SNP loci significantly associated with nutritional elements (potassium, magnesium, calcium, iron, zinc, manganese, copper, and nickel) in sweet maize. Combined with SNPs identified from resequencing data, 20,000 SNPs evenly distributed across the genome were selected, resulting in a combination of 21,768 SNP molecular marker loci shown in Table 2. These combinations are closely linked to the nutritional element content of sweet maize and can be used to analyze the nutritional element content of sweet maize, conduct genome-wide selection breeding for nutritional element traits, and screen for high-quality sweet maize with optimal nutritional elements. The results of the examples show that, based on the SNP molecular marker combinations provided by this invention, the prediction accuracies for the content of nutritional elements K, Mg, Ca, Fe, Zn, Mn, Cu, and Ni are 0.6632, 0.5732, 0.8085, 0.7383, 0.7715, 0.8576, 0.7803, and 0.5922, respectively, which can meet the requirements for genome-wide selection breeding of sweet maize.

[0016] Furthermore, this invention designs probes based on the aforementioned SNP molecular marker combinations and constructs a whole-genome chip, which can be used for genetic diversity and variety analysis of sweet maize. By simply using the chip to identify the genotype of sweet maize inbred lines, the nutrient element content of these lines can be predicted. This method has advantages such as low cost, clear selection targets, early detection, and immunity to environmental influences. The sweet maize whole-genome chip disclosed in this invention can also be used for breeding programs that aggregate superior traits in sweet maize, which is of great significance for improving the efficiency of sweet maize breeding. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The distribution of BLUP values ​​for eight nutrients in 477 fresh corn kernels; Figure 2 The inverse normal transformation results of BLUP values ​​of eight nutrients in 477 fresh corn kernels; Figure 3 PCA analysis results for 477 fresh sweet corn populations; Figure 4 Manhattan plot of GWAS analysis results of 8 element content traits in 477 fresh corn kernels; Figure 5 QQ plot of GWAS analysis results of 8 element content traits in 477 fresh corn kernels; Figure 6 The number and density of SNP molecular marker sites of the present invention on the ten chromosomes of maize; Figure 7The prediction accuracy of the 20K fresh corn breeding chip developed for this invention in whole-genome selection of 8 nutrients. Detailed Implementation

[0019] This invention provides a combination of SNP molecular markers related to the nutritional traits of fresh maize. The positions of the SNP molecular marker combination on the maize reference genome B73 RefGen_v4 version are shown in Table 2 of the specification.

[0020] This invention, through genome-wide association study (GWAS), screened 257 SNP loci significantly associated with eight nutritional elements (K, Mg, Ca, Fe, Zn, Mn, Cu, and Ni) in sweet maize. Combined with SNPs identified by resequencing data, 20,000 SNPs evenly distributed across the genome were selected, resulting in the 21,768 SNP molecular marker loci combinations shown in Table 2. The SNP molecular marker loci provided by this invention have the most markers on chromosome 1 (3,192) and the fewest on chromosome 10 (1,500), showing a positive correlation with chromosome length. These can be used for genetic diversity and variety analysis in sweet maize, analyzing the content of the eight nutritional elements in sweet maize, conducting genome-wide selection breeding for the eight nutritional element traits in sweet maize, and screening for sweet maize with high-quality nutritional elements.

[0021] This invention provides a probe for detecting the SNP molecular marker combinations described in the above-mentioned technical solutions.

[0022] In one implementation, the probes of this invention are synthesized based on the location of the SNP site and the 200 bp sequences flanking it, with a GC content between 40% and 60%, a TM value ≥ 65°C, a sequence complexity LCC value ≥ 0.9, and a number of identical base repeats ≤ 6. The number of homologous regions of the probe sequence across the entire genome is ≤ 3. This invention utilizes a dual-probe design based on the GBTS marker platform, ensuring the accuracy of the sequencing sites.

[0023] This invention provides a chip related to the nutritional characteristics of fresh corn, the chip being used to detect the molecular marker combinations described in the above technical solution.

[0024] In one embodiment, the chip of the present invention is a liquid phase chip. In another embodiment, the chip of the present invention includes the probes described in the above-described technical solution.

[0025] This invention provides a whole genome sequencing kit for fresh corn, comprising the chip described in the above technical solution.

[0026] As one embodiment, the fresh corn whole genome detection kit of the present invention also includes a genomic DNA extraction reagent.

[0027] The present invention provides the application of reagents for detecting SNP molecular marker combinations described in the above technical solutions, or probes described in the above technical solutions, or chips described in the above technical solutions, or whole genome detection kits for fresh corn described in the above technical solutions in one or more of the following: (1) analysis of target traits of fresh corn; the target traits include nutritional elements; (2) breeding and / or assisted breeding of fresh corn with high-quality nutritional elements; (3) analysis of genetic diversity of fresh corn; (4) analysis of fresh corn varieties.

[0028] In one embodiment, the target trait analysis of fresh sweet corn according to the present invention includes predicting the nutrient element content of fresh sweet corn. In one embodiment, the nutrient elements mentioned in the present invention include one or more of potassium, magnesium, calcium, iron, zinc, manganese, copper, and nickel. In one embodiment, the nutrient elements mentioned in the present invention are the nutrient elements of fresh sweet corn kernels.

[0029] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a combination of SNP molecular markers and a chip related to the nutritional traits of fresh corn, as well as their applications. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 1. Identification of 8 nutritional elements in natural populations of fresh corn Forty-seventy-seven fresh-eating maize inbred lines (170 sweet maize, 286 waxy maize, and 21 sweet waxy maize) were planted for three consecutive years at the Zhuangxing Experimental Base of the Shanghai Academy of Agricultural Sciences and self-pollinated. After the maize ears matured, they were threshed and dried. Three ears from each inbred line were selected, and 2g samples from each ear were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) for eight nutrients (potassium (K), magnesium (Mg), calcium (Ca), iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), and nickel (Ni)). After the analysis, the best linear unbiased predictive value (BLUP) of the phenotype was calculated using Meta-R software. The results are as follows: Figure 1 As shown in Table 1, this provides high-quality phenotypic data for subsequent genome-wide association analysis and genome-wide selection analysis.

[0031] Table 1. Content of 8 nutrients in 477 fresh corn samples (mg / kg) 2. Genotyping DNA was extracted from the young leaves of 477 maize inbred lines using the CTAB method. After passing quality testing, the DNA was sent to Novogene Co., Ltd., which then performed whole-genome resequencing at a preset depth of 5× using the Illumina sequencing platform.

[0032] After obtaining the raw sequencing data, variant identification analysis was performed. The specific steps are as follows: (1) Use the FASTP software to perform quality filtering based on the Q20 standard; (2) Use the MEM algorithm of the BWA software to align the filtered sequences to the maize reference genome b73 (version: v4, download address: https: / / download.maizegdb.org / Zm-B73-REFERENCE-GRAMENE-4.0 / Zm-B73-REFERENCE-GRAMENE-4.0.fa.gz); (3) Use the PICARD software to remove PCR repetitive sequences in the sequencing data; (4) Finally, use the Freebayes software to identify SNP variants.

[0033] After obtaining the original variant sites, bcftools and plink software were used for filtering. The filtering criteria were: (1) only biallelic SNP sites were retained; (2) SNP sites with a minor allele frequency greater than 0.5 were retained; (3) SNP sites with a sequencing depth between 2× and 50× and a quality greater than 30 were retained; (4) SNP sites with a sample missing rate less than 0.1 and a site missing rate less than 0.1 were retained. At the same time, in order to reduce linkage disequilibrium redundancy between markers, PLINK was used to perform LD processing on SNPs before genome-wide association analysis. The sliding window method was adopted, with a window size of 50 SNPs and a step size of 5 SNPs. When the linkage disequilibrium coefficient r of any two SNPs in the window was 0. 2 When the value is greater than 0.2, only one site is retained. After filtering, a total of 1,266,886 high-quality SNP sites were obtained.

[0034] 3. Genome-wide association analysis Genome-wide association analysis was performed using GEMMA software. A mixed linear model was employed, and the top three principal components of the PCA analysis were used as the population structure matrix. Phylogenetic relationships were incorporated into the analysis, and a p-value threshold of 10 was used. -5 1e-5, ultimately yielding 257 SNP loci and 8 significant associations with nutrient elements ( Figures 3-5 ).

[0035] 4. Development of liquid phase capture breeding chips Based on the 257 SNPs significantly associated with 8 nutrient elements obtained in step 3, 1,266,886 high-quality SNP loci obtained in step 2 were randomly selected to supplement the 20K markers for probe design on the GBTS marker platform. To ensure the accuracy of sequencing sites, a dual-probe design was adopted (design principles: TM ≥ 65℃, number of homology regions ≤ 3, ambiguous bases 0, sequence complexity LCC value ≥ 0.9, number of identical base repeats ≤ 6, number of tandem repeats ≤ 3) for the development of breeding for sweet corn. Finally, probes for 21,768 loci were designed and synthesized for subsequent genotyping analysis. The physical locations of SNP molecular markers in the 20K sweet corn breeding chip were referenced from the B73 RefGen_v4 version of the maize inbred line B73. Specific location information and their distribution on chromosomes are shown in Table 2 and [Table data missing]. Figure 6 As shown.

[0036] Table 2. Physical location information of SNP molecular markers in 20K fresh corn breeding chips. According to Table 2 and Figure 6 It can be seen that the most SNP molecular markers of 21,768 are on chromosome 1 (3,192), while the fewest are on chromosome 10 (1,500), which is positively correlated with chromosome length.

[0037] 5. Validation of its effectiveness in genome-assisted breeding To confirm the effectiveness of the developed 20K sweet corn breeding chip in genome-wide selection breeding of sweet corn, genome-wide selection was applied to 381 sweet corn accessions (192 sweet corn accessions, 168 waxy corn accessions, and 21 sweet waxy corn accessions). A prediction model was constructed using the rrblup package in R, and five-fold cross-validation was used to calculate the model's prediction accuracy. The prediction accuracy was used to evaluate the effectiveness of the developed 20K sweet corn breeding chip in sweet corn breeding. The results are as follows: Figure 7 As shown, the prediction accuracies of the prediction models for K, Mg, Ca, Fe, Zn, Mn, Cu, and Ni are 0.6632, 0.5732, 0.8085, 0.7383, 0.7715, 0.8576, 0.7803, and 0.5922, respectively. This result indicates that the 20K fresh maize breeding chip can meet the requirements of whole-genome selection breeding for fresh maize.

[0038] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A combination of SNP molecular markers related to nutritional traits of fresh corn, characterized in that, The positions of the SNP molecular marker combinations on the maize reference genome B73 RefGen_v4 version are shown in Table 2 of the specification.

2. A probe for detecting the SNP molecular marker combination as described in claim 1.

3. The probe according to claim 2, characterized in that, The probe is synthesized based on the location of the SNP site and the 200 bp sequence flanking it, with a GC content between 40% and 60%, a TM value ≥ 65℃, a sequence complexity LCC value ≥ 0.9, a number of identical base repeats ≤ 6, and the number of homologous regions of the probe sequence in the whole genome ≤ 3.

4. A chip related to the nutritional characteristics of fresh corn, characterized in that, The chip is used to detect the molecular marker combination as described in claim 1.

5. The chip according to claim 4, characterized in that, The chip is a liquid phase chip.

6. The chip according to claim 4 or 5, characterized in that, The chip includes the probe as described in claim 2.

7. A whole genome sequencing kit for fresh corn, characterized in that, Includes the chip described in any one of claims 4 to 6.

8. The use of the reagent for detecting the SNP molecular marker combination of claim 1, or the probe of claim 2 or 3, or the chip of any one of claims 4 to 6, or the fresh maize whole genome detection kit of claim 7, in one or more of the following: (1) Analysis of target traits of fresh corn; the target traits include nutritional elements; (2) Breeding and / or assisted breeding of high-quality nutrient-rich fresh corn; (3) Analysis of genetic diversity in fresh maize; (4) Analysis of fresh corn varieties.

9. The application according to claim 8, characterized in that, The target trait analysis of fresh corn includes the prediction of the nutrient content of fresh corn.

10. The application according to claim 8 or 9, characterized in that, The nutrients include one or more of potassium, magnesium, calcium, iron, zinc, manganese, copper, and nickel.

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