Development and application of molecular marker for main-effect qlyi1.1 of maize seedling stage tolerance to waterlogging
Patent Information
- Application Number
- CN202610848056.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-12
- Publication Date
- 2026-08-21
AI Technical Summary
[0005]叶片的健康程度是植株健康与否的重要表征，对不同基因型的玉米材料而言，渍水条件下叶片发黄的程度表现出很大的差异，但是这种适应渍水胁迫的位点信息及调控机制，在玉米中还不是很清楚
[0028]本发明首次基于全基因组关联分析对一个新的控制玉米苗期渍水叶黄指数主效QTL进行了精细定位，将该主效QTL定位于1号染色体，该区间包含3个显著的SNP位点，总长度为700kb（Chr1：206312223-207012223），该位点均为控制耐渍表型变异的主效QTL位点，与之紧密连锁的优良单倍型SNP标记，位于玉米B73参考基因组(Zm-B73-REFERENCE-NAM-5.0)第一染色体的第206662223碱基处，解释的渍水叶黄指数表型贡献率为6.16%，基于其最佳等位基因型开发的PARMS标记可用于分子标记辅助选择育种。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology, specifically relating to the acquisition of the main QTL qLYI1.1 for waterlogging tolerance in maize seedlings, and the development and application of its molecular marker primers. Background Technology
[0002] With the intensification of climate anomalies and uneven rainfall during the maize growing season, waterlogging stress has become one of the major natural disasters facing maize production (Zhu Shidie et al., 2024). Developing waterlogging-tolerant varieties with high yield potential is one of the main goals of maize breeding. Understanding the mechanisms of waterlogging tolerance is of great significance for breeders to develop new waterlogging-tolerant varieties with different waterlogging tolerance traits (Zaidi et al., 2004).
[0003] The severity of waterlogging damage is related to the duration of stress, soil conditions, climate, cultivation techniques, crop growth stage, and genotype (Mano et al., 2002). Studies have shown that waterlogging during the seedling stage of maize for 6 days is more severe than during the jointing and tasseling stages (Ren et al., 2023), especially from the second leaf stage (V2) to the seventh leaf stage (V7), where the root system is the first to be affected by waterlogging stress (Zaidi et al., 2004). After 6 days of waterlogging treatment, most roots, except for some adventitious roots, tend to rot, and the plants cannot absorb soil nutrients well, resulting in nitrogen deficiency, leaching, and denitrification, the latter manifesting as leaf yellowing (Ren et al., 2017). Root hypoxia leads to decreased respiration rate and root vigor, accelerating leaf senescence and reducing indicators such as photosynthetic rate, intercellular CO2 concentration, transpiration rate, and stomatal conductance. Photosynthetic indicators, such as net photosynthetic rate, decrease with prolonged flooding, and the magnitude of the decrease is directly proportional to the duration of flooding (Zhou Qingyun et al., 2020; Zhang Wei et al., 2017; Han Liangliang et al., 2011). Flooding can disrupt chlorophyll synthesis; 30 days of flooding inhibits chlorophyll a synthesis, affecting photosynthesis (Zhang Wei et al., 2017), resulting in severe energy deficiency (Kaur et al., 2020; Tian et al., 2021). Maize cannot withstand low-oxygen conditions in the root zone, leading to a significant reduction in yield (Dennis et al., 2000).
[0004] However, the current scarcity of waterlogging-tolerant maize germplasm resources, insufficient discovery of key genes, unclear molecular regulatory mechanisms, and lack of efficient molecular breeding technologies severely restrict the cultivation of new waterlogging-tolerant varieties. Discovering and effectively utilizing waterlogging-tolerant genes, exploring the molecular mechanisms of waterlogging tolerance in maize, creating new waterlogging-tolerant maize germplasm, and cultivating new waterlogging-tolerant varieties are the most economical and effective ways to reduce maize losses, increase yield per unit area, and expand the maize planting area (Zaidi et al., 2004, 2010).
[0005] The health of leaves is an important indicator of plant health. The degree of yellowing of leaves under waterlogging conditions varies greatly among maize materials of different genotypes. However, the site information and regulatory mechanism of this adaptation to waterlogging stress are not yet clear in maize.
[0006] Based on this, this study extensively collected 567 backbone inbred lines from maize producing areas in Southwest China and the Huang-Huai-Hai Plain, established a population of superior germplasm with different characteristics, completed whole-genome resequencing at a depth of 20X using the DNBSEQ-T7 / PE150 sequencing platform, and evaluated waterlogging at the seedling stage. Furthermore, through association analysis, superior allelic variations were identified, specific functional markers were developed and used to create intermediate breeding materials, providing superior allelic resources for carrying out molecular breeding for maize waterlogging tolerance. Summary of the Invention
[0007] The purpose of this invention is to provide a reagent for detecting the base at position 206662223 of the first chromosome of the maize genome and its application in the screening and breeding of waterlogging tolerance traits in maize seedlings.
[0008] Another objective of this invention is to provide the application of a reagent for detecting the base at position 206662223 of the first chromosome of the maize genome in the preparation of a screening kit for waterlogging tolerance in maize seedlings.
[0009] The final objective of this invention is to provide a method for screening and breeding maize seedlings to tolerate waterlogging.
[0010] To achieve the above objectives, the present invention is implemented through the following technical solution:
[0011] Obtaining the main-effect QTL qLYI1.1 for controlling the yellowing index of maize seedlings due to waterlogging:
[0012] 1) Through genome-wide association analysis, this invention detected the QTL qLYI1.1, which regulates the yellowing index of seedling waterlogging, on chromosome 1 of maize. This region contains 3 significant SNP sites with a total length of 700kb (Chr1: 206312223-207012223). Furthermore, a superior haplotype SNP marker closely linked to it was discovered, located at base 206662223 on chromosome 1 of the maize B73 reference genome (Zm-B73-REFERENCE-NAM-5.0), which explained 6.16% of the yellowing index phenotype contribution.
[0013] 2) For the above-mentioned SNP molecular markers, the applicant has developed PARMS marker primers, which are as follows:
[0014] qLYI1.1F1 (Waterlogging Resistance Haplotype A): GAAGGTGACCAAGTTCATGCTTCTGAATCGATTAATACCCATCTGA;
[0015] qLYI1.1F2 (Water-sensitive haplotype G): GAAGGTCGGAGTCAACGGATTTCTGAATCGATTAATACCCATCTGG;
[0016] And qLYI1.1R:CCCATTCTGGTCTTGAGAAACCTA.
[0017] The scope of protection of this invention also includes:
[0018] Application of reagents for detecting base position 206662223 on chromosome 1 of maize genome in screening breeding for waterlogging tolerance during maize seedling stage.
[0019] Application of reagent for detecting base position 206662223 on chromosome 1 of maize genome in the preparation of a screening kit for waterlogging tolerance in maize seedlings.
[0020] In the above-described applications, preferably, the reagent is a primer.
[0021] In the above-described applications, if a homozygote is detected with a base A at position 206662223 on the first chromosome of the maize genome, the maize is determined to be waterlogged tolerant maize during the seedling stage. If a homozygote is detected with a base G at position 206662223 on the first chromosome of the maize genome, the maize is determined to be waterlogged sensitive maize during the seedling stage.
[0022] In the above-described applications, the yellowing index of the seedling-tolerant corn is less than 1.00, while the yellowing index of the seedling-sensitive corn is greater than 1.50.
[0023] The primers described above are preferably PARMS detection primers, and more preferably the primers provided by this invention: qLYI1.1F1 (waterlogging resistant haplotype A): GAAGGTGACCAAGTTCATGCTTCTGAATCGATTAATACCCATCTGA; qLYI1.1F2 (waterlogging sensitive haplotype G): GAAGGTCGGAGTCAACGGATTTCTGAATCGATTAATACCCATCTGG; and qLYI1.1R: CCCATTCTGGTCTTGAGAAACCTA.
[0024] The above-described applications, including waterlogging tolerance traits such as leaf yellowing index, survival rate, seedling fresh weight, and seedling dry weight, are described.
[0025] A method for screening and breeding maize seedlings for waterlogging tolerance includes detecting the 206662223rd base on chromosome 1 of the maize genome using conventional methods in the art. These conventional methods include, but are not limited to, sequencing, TaqMan probe method, AS-PCR method, molecular beacon method, high-resolution melting curve method, CAPS method, SNaPshot method, KASP method, PARMS method, gene chip method, or mass spectrometry method.
[0026] The reference genome of maize used in this invention is Zm-B73-REFERENCE-NAM-5.0.
[0027] Compared with the prior art, the present invention has the following advantages:
[0028] This invention is the first to finely map a novel major QTL controlling waterlogging-induced yellowing index in maize seedlings based on genome-wide association analysis. The major QTL is located on chromosome 1, which contains three significant SNP loci with a total length of 700 kb (Chr1: 206312223-207012223). These loci are all major QTL loci controlling waterlogging tolerance phenotype variations. The superior haplotype SNP marker closely linked to it is located at base 206662223 on chromosome 1 of the maize B73 reference genome (Zm-B73-REFERENCE-NAM-5.0), explaining 6.16% of the waterlogging-induced yellowing index phenotype. The PARMS marker developed based on its optimal allele can be used for marker-assisted selection breeding.
[0029] The applicant verified that the superior haplotype qLYI1.1 can reduce the yellowing index by 41.69% in natural populations by using the yellowing index phenotype of seedling waterlogging in 567 maize inbred lines. Moreover, it has a good selection effect on traits such as survival rate, seedling fresh weight, and seedling dry weight under maize waterlogging stress. This superior haplotype provides genetic resources for the creation of waterlogging-resistant maize lines. Attached Figure Description
[0030] Figure 1 Partial field photos of the seedling waterlogging tolerance assessment of 567 inbred line materials;
[0031] The control group had 3 replicates, and the waterlogging treatment group had 3 replicates.
[0032] Figure 2 The distribution of leaf chlorosis index in an inbred line population after two weeks of waterlogging.
[0033] Figure 3 This is a schematic diagram of the qLYI1.1 site association analysis;
[0034] Association analysis of 13.2 million polymorphic variation sites with a minimum allele frequency greater than 0.05 at the qLYI1.1 locus with the waterlogged leaf yellowing index phenotype in 567 different inbred lines, with each dot representing a polymorphic site.
[0035] Figure 4 A schematic diagram for the analysis of superior haplotype effects;
[0036] Comparative analysis of leaf yellow index of 45 Hap1 inbred lines and 469 Hap2 inbred lines was conducted. Each box represents the median and interquartile range, extended to the maximum and minimum values. The significance of the differences was estimated by one-way ANOVA.
[0037] Figure 5 This is a schematic diagram illustrating the genetic effects of qLYI1.1 on traits such as survival rate, seedling fresh weight, and seedling dry weight waterlogging tolerance coefficient.
[0038] Scatter plots represent the distribution of family survival rate, seedling fresh weight, and seedling dry weight waterlogging tolerance coefficient. Each box represents the median and interquartile range, extended to the maximum and minimum values. Error bars represent SD. The significance of differences was estimated by one-way ANOVA.
[0039] Figure 6 A schematic diagram illustrating the development and utilization of the optimal haplotype functional marker for qLYI1.1;
[0040] In the diagram: Blue: qLYI1.1 type families have strong resistance to waterlogging; Green: qlyi1.1 type families have weak resistance to waterlogging. Detailed Implementation
[0041] Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the field; unless otherwise specified, the reagents or materials described are all publicly available.
[0042] The reference genome for maize in this invention is Zm-B73-REFERENCE-NAM-5.0 (MaizeGDB GenomeCenter).
[0043] Example 1:
[0044] Obtaining the QTL qLYI1.1 for waterlogging tolerance in maize seedlings:
[0045] 1. Materials and Methods
[0046] 1.1 Materials
[0047] A total of 567 backbone inbred lines from maize producing areas in Southwest China and the Huang-Huai-Hai Plain were collected. A population of superior germplasm with different characteristics was established. The whole genome was resequencing at a depth of 20X based on the DNBSEQ-T7 / PE150 sequencing platform, and 13.2 million high-quality SNP markers were obtained.
[0048] 1.2 Experimental Methods
[0049] 1.2.1 Phenotypic Identification
[0050] For related populations, 20 seeds from each family were planted in a rectangular plastic box (60 cm long, 25 cm deep, 35 cm wide) divided into 10 compartments, each containing 30 kg of quartz sand. Each experiment was repeated three times, with no replicates for each square box. At the two-leaf stage, 10 uniformly growing seedlings were retained from each replicate and subjected to waterlogging, with the water level maintained 5-7 cm above the quartz sand. A parallel control experiment under normal growth conditions was also conducted. After 8 days of waterlogging stress, 10 traits of seedlings under control (CK) and waterlogging (WT / R) conditions were measured, including seedling height (cm), root length (cm), seedling fresh weight (g), root fresh weight (g), seedling dry weight (g), root dry weight (g), number of adventitious roots, number of primary roots, and total number of roots (adventitious roots + primary roots). The yellowing index and survival rate were scored only under waterlogging conditions. If seedlings did not show leaf chlorosis, the yellowing index was 0; if 50% of the first leaf was chlorotic, the yellowing index was 0.5, and so on. The survival rate was the ratio of the number of surviving seedlings to the total number of seedlings, and the waterlogging tolerance coefficient was the ratio of the trait value under waterlogging conditions to the trait value under control conditions.
[0051] 1.2.1 Genome-wide association analysis of the leaf yellowing index loci during seedling waterlogging
[0052] For the raw sequencing data after sequencing, data quality control was performed to obtain high-quality clean data. This clean data was then aligned to a reference genome for variant detection. The reference genome used was AGPv5, downloaded from http: / / plants.ensembl.org / Zea_mays / Info / Index. BWA software was used to align PE reads with the B73 reference genome sequence, obtaining alignment results in AM format. The SAM format file was converted to BAM format using samtools software. Then, the reads in the BAM file were sorted using the SortSam tool in Picard, and PCR duplicates were removed to obtain the final BAM file suitable for variant calling. Finally, the HaplotypeCaller module of GATK was used for variant detection, including SNPs and InDels. Q and K were calculated using STRUCTURE and TESSEL 5.0 software, respectively. After correction, the P-value was set to 1.0 × 10⁻⁶. -5 As a threshold for the significance of GWAS results.
[0053] 1.2.2 Development of optimal haplotype molecular markers
[0054] Based on the B73 genome and the differential site information provided by qLYI1.1 sequencing, specific primers were designed. Using PARMS detection technology, the adapter sequence that matches FAM fluorescence is GAAGGTGACCAAGTTCATGCT, and the adapter sequence that matches HEX fluorescence is GAAGGTCGGAGTCAACGGATT.
[0055] 1.2.3 Genotype Analysis
[0056] Small-scale DNA extraction from maize was performed using the CTAB (Cetyltrimethyl Ammonium Bromide) method (Saghai-Maroof et al 1984), followed by PARMS SNP detection.
[0057] 2. Results and Analysis
[0058] 2.1 Evaluation of the waterlogging resistance of 567 inbred lines
[0059] At the two-leaf-one-heart stage, the plants were treated with waterlogging for two weeks. The control group grew normally. Among the population groups, the root length waterlogging tolerance coefficient was the lowest at 0.48, with a variation range of 0.17-0.99. There were highly significant differences in root length between the control group and the waterlogging treatment group. The waterlogging tolerance coefficient for fresh root weight was 0.63, with a variation range of 0.12-0.98, and the waterlogging tolerance coefficient for dry root weight was 0.76, with a variation range of 0.20-1.03. This indicates that there is relatively rich genetic variation among the populations, and root length is the most sensitive to waterlogging stress. The waterlogging tolerance coefficients for indeterminate root number and total root number were 1.06 and 1.04, respectively, indicating that waterlogging stress has a relatively small impact on the root coefficient (Table 1).
[0060] The population was divided into 6 levels based on the yellowing index of leaves after 2 weeks of waterlogging. Among them, there were 127 inbred lines with a yellowing index greater than 2, 168 inbred lines with a yellowing index less than 1, and 272 inbred lines with a yellowing index between 1 and 2. Figure 2 ).
[0061] Table 1. Evaluation of the waterlogging tolerance of inbred line populations
[0062] .
[0063] WT represents the waterlogged treatment group, CK represents the normal growth group, and T-test two-way ANOVA is used for significance testing.
[0064] 2.2 Identification and genetic effect analysis of the qLYI1.1 gene locus for the yellowing leaf index during the seedling stage.
[0065] This application identifies a novel major-effect QTL controlling the yellow leaf index in maize seedlings based on genome-wide association analysis. This major-effect QTL is located on chromosome 1, and the region contains three significant SNP loci with a total length of 700 kb (Chr1: 206312223-207012223), named qLYI1.1. Figure 3 ).
[0066] qLYI1.1 locus lead SNP 206662223 (A / G) A significant association was found at P=4.49E-06, located at base 206662223 on chromosome 1 of the maize genome (maize B73 reference genome Zm-B73-REFERENCE-NAM-5.0, referred to in this invention as maize B73V5 reference genome), explaining 6.16% of the contribution of the waterlogged leaf yellowing index phenotype.
[0067] PARMS primers were designed for the above SNP sites as follows:
[0068] (1) Label the leadSNP for peak SNPs closely linked to qLYI1.1 206662223 (A / G)The sequence of 200 bp upstream and downstream of position 206662223 on chromosome 1 of the maize B73V5 reference genome was extracted. The PARMS marker detection primer sequences were obtained according to primer design principles as follows:
[0069] qLYI1.1F1: GAAGGTGACCAAGTTCATGCT TCTGAATCGATTAATACCCATCTGA, as shown in SEQ ID NO.3;
[0070] qLYI1.1F2: GAAGGTCGGAGTCAACGGATT TCTGAATCGATTAATACCCATCTGG, as shown in SEQ ID NO.4;
[0071] qLYI1.1R: CCCATTCTGGTCTTGAGAAACCTA, as shown in SEQ ID NO.5;
[0072] The underlined part of the forward primer is the fluorescent adapter sequence.
[0073] (2) Using the genomic DNA of the maize inbred line population as a template, the above primers were used to perform real-time PCR amplification. The FAM and HEX signals were scanned using a Tecan F200 and the results were output. Finally, the genotype was converted.
[0074] Using the primers described above, the sequence amplified in FL310 (the male parent of Kangnong 2) is: TCTGAATCGATTAATACCCATCTG A GGATCTCCAACCACACGACATATCTAGCACTTAACCCTTGCATATGTCAACCCACACCTAGGTTTCTCAAGACCAGAATGGG, as shown in SEQ ID NO.1;
[0075] The sequence amplified in the stain-sensitive material 568G (Suyu 15 parent) is: TCTGAATCGATTAATACCCATCTG G GGATCTCCAACCACACGACATATCTAGCACTTAACCCTTGCATATGTCAACCCACACCTAGGTTTCTCAAGACCAGAATGGG, as shown in SEQ ID NO.2.
[0076] Amplification system:
[0077] .
[0078] Amplification parameters:
[0079] .
[0080] According to SNP 206662223(A / G) The 567 maize inbred lines were divided into two haplotypes, named Hap1 and Hap2, and then paired to compare the differences in leaf chlorosis index between the two haplotypes after two weeks of waterlogging. Among these, 45 inbred lines were SNPs. 206662223(A / A) Also known as the Hap1 allele, 469 materials were SNPs. 206662223(G / G) Alternatively referred to as the Hap2 allele, other heterozygous sites were filtered out and not counted. Compared to the Hap2 haplotype, the leaf yellowing index of the Hap1 haplotype inbred lines was reduced by an average of 41.69% (p=1.21E-08). Figure 4 Therefore, Hap1 is a superior haplotype of qLYI1.1, accounting for 8.8%.
[0081] Meanwhile, qLYI1.1 is a pleiotropic gene locus. Under waterlogging stress, its superior haplotype Hap1 significantly improved survival rate, seedling fresh weight, and seedling dry weight waterlogging tolerance coefficient. Compared with the Hap2 haplotype, the survival rate increased by an average of 0.19 (p=9.38E-05), the seedling fresh weight waterlogging tolerance coefficient increased by an average of 0.08 (p=0.012), and the seedling dry weight waterlogging tolerance coefficient increased by an average of 0.09 (p=0.016). Figure 5 Furthermore, Hap1 accounts for only 8.8% of the material, suggesting that there is still considerable room for improvement in the waterlogging resistance of inbred lines using qLYI1.1 in breeding practices.
[0082] Example 2:
[0083] Application of the superior haplotype molecular marker primer qLYI1.1, a major QTL for waterlogging tolerance in maize seedlings:
[0084] In 214 maize inbred lines and 1200 local germplasm populations, after one week of waterlogging at the two-leaf-one-heart stage, 10 families with a yellow leaf index (YLE) less than 1.00 and 10 families with a YLE greater than 1.50 were randomly selected. Eight plants from each family were pooled, and DNA was extracted. Genotyping was performed using PARMS primers developed for the optimal allele at the qLYI1.1 locus in Example 1. The results showed that among the families with a YLE less than 1.00, 8 belonged to the Hap1 allele, and 2 contained the unfavorable Hap2 allele. In contrast, all 10 families with a YLE greater than 1.50 contained the unfavorable Hap2 allele (Table 2). Figure 6 These results confirm that the developed functional markers can be used for molecular marker-assisted selection in the genetic improvement of waterlogging-resistant lines, providing selection targets for creating new waterlogging-resistant maize germplasm and breeding new waterlogging-resistant varieties.
[0085] Table 2 shows that the PARMS designation qLYI1.1 can be used for stain resistance assessment.
[0086] .
[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. The application of a reagent for detecting base position 206662223 on chromosome 1 of the maize genome in the screening and breeding of waterlogging tolerance traits in maize seedlings, characterized in that... If a homozygote is found to have an A base at position 206662223 on chromosome 1 of the maize genome, the maize is determined to be a seedling-tolerant maize. If a homozygote is found to have a G base at position 206662223 on chromosome 1 of the maize genome, the maize is determined to be a seedling-sensitive maize. The maize genome is Zm-B73-REFERENCE-NAM-5.
0.
2. The application of a reagent for detecting base position 206662223 on chromosome 1 of the maize genome in the preparation of a screening kit for waterlogging tolerance in maize seedlings, characterized in that... If a homozygote is found to have an A base at position 206662223 on chromosome 1 of the maize genome, the maize is determined to be a seedling-tolerant maize. If a homozygote is found to have a G base at position 206662223 on chromosome 1 of the maize genome, the maize is determined to be a seedling-sensitive maize. The maize genome is Zm-B73-REFERENCE-NAM-5.
0.
3. The application according to claim 1 or claim 2, characterized in that, The yellowing index of the seedling-tolerant corn is less than 1.00, while the yellowing index of the seedling-sensitive corn is greater than 1.
50.
4. The application according to claim 1 or 2, characterized in that, The reagent mentioned is a primer.
5. The application according to claim 4, wherein the primers are: qLYI1.1F1: GAAGGTGACCAAGTTCATGCTTCTGAATCGATTAATACCCATCTGA, qLYI1.1F2: GAAGGTCGGAGTCAACGGATTTCTGAATCGATTAATACCCATCTGG and qLYI1.1R: CCCATTCTGGTCTTGAGAAACCTA.
6. A method for screening and breeding maize seedlings for waterlogging tolerance, comprising detecting the 206662223rd base of the first chromosome of the maize genome, wherein the method is: sequencing, TaqMan probe method, AS-PCR method, molecular beacon method, high-resolution melting curve method, CAPS method, SNaPshot method, KASP method, PARMS method, gene chip method, or mass spectrometry method. If a homozygote is detected at the 206662223rd base of the first chromosome of the maize genome with a base of A, the maize is determined to be waterlogging tolerant maize at the seedling stage; if a homozygote is detected at the 206662223rd base of the first chromosome of the maize genome with a base of G, the maize is determined to be waterlogging sensitive maize at the seedling stage. The maize genome is Zm-B73-REFERENCE-NAM-5.
0.
7. The application according to claim 1 or 2, or the method according to claim 6, wherein the waterlogging resistance characteristics include leaf yellowing index, survival rate, seedling fresh weight, and seedling dry weight characteristics.