A haplotype molecular marker associated with manganese absorption in maize and its application

Abstract

This invention belongs to the field of molecular genetic marker technology, specifically disclosing a haplotype molecular marker related to manganese absorption in maize and its application. This marker, based on B73 RefGen_v5, consists of two closely linked SNPs at position 176424559 (A / -) and position 176424565 (T / C) on chromosome 8, forming AT and -C haplotypes. The AT haplotype shows a significant positive correlation with grain manganese content, while the -C haplotype shows a significant negative correlation. This marker can be directly applied to genotyping in maize seedlings or early hybrid generations, eliminating the need for field identification of manganese deficiency stress phenotypes. This significantly shortens the breeding cycle and reduces environmental interference, enabling early and precise selection of materials with high grain manganese accumulation.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetic marker technology, specifically relating to a haplotype molecular marker related to manganese absorption in maize and its application. Background Technology

[0002] Manganese is an essential micronutrient for plant growth and development, playing a crucial role in chlorophyll synthesis, photosynthesis, enzyme activation, and stress resistance regulation in maize. Manganese deficiency leads to symptoms such as yellowing leaves, stunted growth, and slow development in maize, severely impacting yield and quality. Currently, soil manganese deficiency has become a significant factor limiting maize production, particularly prevalent in calcareous and high-pH soils.

[0003] With the development of biotechnology and genomics, molecular breeding has become an important research direction in maize breeding. In the field of marker-assisted breeding of maize, researchers have identified a large number of QTLs affecting traits such as yield, agronomy, quality, and biotic and abiotic resistance. To date, more than 2,000 maize QTLs have been included in maize databases. Some major QTLs have been used for marker-assisted selection, directly serving breeding. However, research on molecular markers related to manganese uptake and utilization efficiency in maize remains relatively lacking. Currently, although some studies have focused on the mechanisms of micronutrient uptake in crops, research on the development and application of haplotype molecular markers for maize manganese uptake-related genes is still relatively rare. Existing marker-assisted breeding technologies for maize mainly focus on traits such as yield, resistance, and quality, while research on marker-assisted selection for traits of efficient nutrient utilization is relatively insufficient. Therefore, developing molecular markers related to manganese uptake in maize has significant application value and promotional significance for breeding new maize varieties with efficient manganese utilization.

[0004] Haplotype analysis, a genotyping method based on the combination of multiple linked SNP loci, can more comprehensively reflect the genetic background of alleles compared to a single SNP marker, demonstrating unique advantages in QTL fine mapping and molecular marker development. Applying haplotype molecular marker technology to the study of manganese absorption traits in maize holds promise for solving the problems of low selection efficiency and long cycles in traditional breeding methods, enabling the rapid breeding of new maize varieties with high manganese utilization efficiency. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention aims to provide SNP sites related to manganese absorption in maize and their associated haplotypes, and to reveal their application in marker-assisted breeding of maize with high manganese utilization trait.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] On the one hand, the present invention provides a haplotype molecular marker associated with manganese absorption in maize, the haplotype molecular marker consisting of the following two SNP sites:

[0008] SNP1: Located at position 176424559 on chromosome 8 of the maize genome, with a variant type of A / -;

[0009] SNP2: Located at position 176424565 on chromosome 8 of the maize genome, with a variant type of T / C;

[0010] The physical location of the SNP sites was determined based on the maize B73 reference genome version V5.0 (B73 RefGen_v5);

[0011] The two SNP sites combine to form two haplotypes: the AT haplotype and the -C haplotype;

[0012] The AT haplotype is positively correlated with the manganese content of corn kernels, while the -C haplotype is negatively correlated with the manganese content of corn kernels.

[0013] On the other hand, the present invention also provides the application of the above-mentioned haplotype molecular markers in the preparation of products for predicting or identifying the manganese content of maize kernels.

[0014] Preferably, the product includes a gene chip, a kit, or a reagent.

[0015] On the other hand, the present invention also provides the application of the above-mentioned haplotype molecular markers in the breeding of maize varieties with high manganese accumulation, the improvement of maize grain quality and / or the identification of maize germplasm resources.

[0016] Preferably, the breeding of maize varieties with high manganese accumulation includes using AT haplotypes as selection markers to perform whole-genome selection or genomic breeding value evaluation.

[0017] Preferably, the improvement of corn kernel quality includes breeding manganese-enriched functional corn varieties.

[0018] On the other hand, the present invention also provides a method for identifying or assisting in the identification of manganese content in corn kernels, comprising the following steps:

[0019] (1) Extract maize genomic DNA to be tested;

[0020] (2) Detect the genotypes at positions 176424559 and 176424565 on chromosome 8 of the maize to be tested;

[0021] (3) The haplotype is determined based on the allele combination of the two loci: when the 176424559th position is A and the 176424565th position is T, it is determined to be the AT haplotype; when the 176424559th position is a deletion and the 176424565th position is C, it is determined to be the -C haplotype.

[0022] (4) Predict the manganese content of corn kernels based on haplotype: AT haplotype indicates high manganese accumulation, and -C haplotype indicates low manganese accumulation.

[0023] Preferably, the detection of the genotypes at positions 176424559 and 176424565 of chromosome 8 of the maize to be tested in step (2) is achieved by direct sequencing, KASP genotyping, or high-throughput chip detection based on specific probes.

[0024] On the other hand, the present invention also provides a maize breeding method, characterized by comprising the following steps: detecting haplotype molecular markers in maize germplasm resources or breeding materials, screening and retaining maize individuals containing the AT haplotype as parents for breeding.

[0025] Preferably, the breeding method includes one or more combinations of hybridization, backcrossing, self-pollination, and recurrent selection.

[0026] The beneficial effects of this invention are as follows: By mining two closely linked functional SNP loci (positions 176424559 and 176424565) on maize chromosome 8 and constructing specific haplotype markers, the positive correlation between the AT haplotype and the manganese content of maize kernels and the negative correlation between the -C haplotype were clearly demonstrated for the first time. This overcomes the limitations of existing single SNP markers, which have weak correlation and poor stability. The use of haplotype combinations significantly improves the explanatory power and predictive accuracy of manganese absorption traits. This marker can be directly applied to genotype screening in maize seedlings or early hybrid generations. This study eliminates the need for field identification of manganese deficiency stress phenotypes, significantly shortening the breeding cycle and reducing environmental interference, enabling early and precise selection of materials with high grain manganese accumulation. Furthermore, the marker, obtained through genome-wide association analysis, exhibits broad genetic background adaptability and can effectively cover superior allelic variations in different maize germplasm resources. It provides an efficient and reliable molecular tool for the targeted improvement of low-manganese tolerant varieties and the precise design breeding of micronutrient-rich maize, holding significant practical value for improving maize nutritional quality, reducing manganese fertilizer application in farmland, and ensuring food security. Attached Figure Description

[0027] Figure 1 GWAS analysis of manganese content in maize kernels from three environmental populations.

[0028] Figure 2Linkage disequilibrium analysis of significant association markers (A) and phenotypic distribution and variance analysis of different haplotypes (B). Detailed Implementation

[0029] The following examples are further illustrations of the present invention, but not limitations thereof. Specific experimental conditions and methods are not specified in the following examples, and the techniques used are generally conventional methods well known to those skilled in the art.

[0030] Example 1: Development of haplotype molecular markers associated with manganese absorption in maize

[0031] 1. Experimental Methods

[0032] (1) Phenotypic determination

[0033] This invention uses 187 maize inbred line related population germplasm resources as experimental materials. Phenotypic identification experiments were conducted in three environments, namely the Hainan Experimental Station (environment 1, E1) and the Jiangmen Experimental Station (environments 2 and 3, E2 and E3), both of which adopted a randomized complete block design. The manganese content of maize kernels was determined by microwave digestion-inductively coupled plasma mass spectrometry.

[0034] (2) Genotyping

[0035] DNA was extracted from 187 maize inbred lines and high-throughput sequencing was performed. After filtering out adapters and low-quality reads, the reads were aligned to the B73 maize reference genome (B73 RefGen_v5) using bwa software. Finally, SNP information was extracted using software to obtain the genotype of each inbred line.

[0036] (3) Genome-wide association analysis (GWAS)

[0037] The kinship matrix among inbred lines was calculated using Tassel5 software. Population structure (K value of 4) was calculated using Admixture software. A GWAS analysis was performed using a mixed linear model (MLM) based on Tassel5, with the population structure Q-value and kinship coefficient as correction factors, to identify Peak SNPs.

[0038] 2. Experimental Results

[0039] The results of manganese content and SNP genotyping data in kernels of 187 maize inbred lines are as follows:

[0040] Genome-wide association studies (GWAS) were conducted in three environments (E1: Hainan, 2013; E2: Jiangmen, Guangdong, 2020; E3: Jiangmen, Guangdong, 2021). For example... Figure 1As shown, two significant features (P < 7.98 × 10⁻⁶) were identified in the 176Mb region of chromosome 8. -8 The SNP markers (with a Bonferroni correction threshold of 0.05 / 626535 [total number of SNPs used for GWAS analysis]) were 8_176424559 (variate type: A / -) and 8_176424565 (variate type: T / C). These two sites were identified in all three environments, and linkage disequilibrium analysis showed they were strongly and tightly linked (R0). 2 = 1.0), belonging to one haplotype block, forming two haplotypes: AT haplotype and -C haplotype ( Figure 2 A).

[0041] Locus effect analysis revealed that haplotype "AT" promoted manganese accumulation in grains, with an average effect value of +0.5558 μg / g across the three environments (E1: +0.8578, E2: +0.4250, E3: +0.3845), making it a superior manganese-efficient haplotype. Conversely, haplotype "-C" was detrimental to manganese accumulation in grains, with an average effect value of -0.6066 μg / g (E1: -0.8446, E2: -0.4857, E3: -0.4895). Analysis of variance showed a highly significant difference between the two haplotypes (P = 0.014). Figure 2 B). This result can be used for early material screening to determine the amount of manganese accumulated in maize kernels, reducing subsequent human and material costs.

[0042] Example 2: Validation of haplotype molecular markers

[0043] Based on the haplotype molecular markers related to manganese absorption in maize developed in this invention (composed of SNP at position 176424559 (A / -) and SNP at position 176424565 (T / C) on maize chromosome 8), 100 maize inbred lines were selected as materials for verification. Analysis of variance was performed on the associated population of these maize inbred lines. The results of haplotype phenotypic effect analysis (Table 1) show that the average synergistic effect of haplotype "AT" was +0.5615 (μg / g), and the average synergistic effect of haplotype "–C" was -0.6123 (μg / g), with significant differences (P=0.017).

[0044] Table 1. Analysis of haplotype phenotypic effect values

[0045] The above results indicate that the “AT” haplotype is a high-manganese-accumulating type and the “–C” haplotype is a low-manganese-accumulating type, with stable and significant differences. The independent verification results of this group show good repeatability, confirming that this haplotype molecular marker can stably distinguish between high and low manganese content in maize kernels. It is suitable for the identification of high-manganese-accumulating maize germplasm, the breeding of manganese-rich varieties, and molecular marker-assisted breeding.

[0046] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A haplotype molecular marker associated with manganese absorption in maize, characterized in that, The haplotype molecular marker consists of the following two SNP sites: SNP1: Located at position 176424559 on chromosome 8 of the maize genome, with a variant type of A / -; SNP2: Located at position 176424565 on chromosome 8 of the maize genome, with a variant type of T / C; The physical location of the SNP sites was determined based on the maize B73 reference genome version V5.0; The two SNP sites combine to form two haplotypes: the AT haplotype and the -C haplotype; The AT haplotype is positively correlated with the manganese content of corn kernels, while the -C haplotype is negatively correlated with the manganese content of corn kernels.

2. The use of the haplotype molecular marker of claim 1 in the preparation of products for predicting or identifying the manganese content of maize kernels.

3. The application according to claim 2, characterized in that, The products include gene chips, kits, or reagents.

4. The application of the haplotype molecular marker as described in claim 1 in the breeding of maize varieties with high manganese accumulation, the improvement of maize grain quality, and / or the identification of maize germplasm resources.

5. The application according to claim 4, characterized in that, The breeding of maize varieties with high manganese accumulation includes using AT haplotypes as selection markers to conduct whole-genome selection or genomic breeding value evaluation.

6. The application according to claim 4, characterized in that, The improvement of corn kernel quality includes the breeding of manganese-enriched functional corn varieties.

7. A method for identifying or assisting in the identification of manganese content in corn kernels, characterized in that, Includes the following steps: (1) Extract maize genomic DNA to be tested; (2) Detect the genotypes at positions 176424559 and 176424565 on chromosome 8 of the maize to be tested; (3) The haplotype is determined based on the allele combination of the two loci: when the 176424559th position is A and the 176424565th position is T, it is determined to be the AT haplotype; when the 176424559th position is a deletion and the 176424565th position is C, it is determined to be the -C haplotype. (4) Predict the manganese content of corn kernels based on haplotype: AT haplotype indicates high manganese accumulation, and -C haplotype indicates low manganese accumulation.

8. The method for identifying or assisting in the identification of manganese content in corn kernels according to claim 7, characterized in that, The detection of the genotypes at positions 176424559 and 176424565 on chromosome 8 of the maize to be tested, as described in step (2), is achieved by direct sequencing, KASP genotyping, or high-throughput chip detection based on specific probes.

9. A method for breeding maize, characterized in that, Includes the following steps: The haplotype molecular markers as described in claim 1 are detected in maize germplasm resources or breeding materials, and maize individuals containing the AT haplotype are screened and retained as parents for breeding.

10. The maize breeding method according to claim 9, characterized in that, The breeding methods include one or more combinations of hybridization, backcrossing, self-crossing, and recurrent selection.

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