Molecular marker, detection reagent closely linked to corn leaf color phase regulation trait and application thereof

Abstract

The application discloses a molecular marker closely linked with a maize leaf color stage regulation trait, a detection reagent and application thereof, and belongs to the technical field of molecular genetics. The application identifies an InDel molecular marker closely linked with a maize seedling stage leaf color stage regulation trait on the 6th chromosome of maize for the first time, and the marker can be used for accurate genotype identification of a maize population in early breeding, and can be used for efficient screening of individuals carrying a leaf color regulation gene. The material gradually appears a white phenotype before V5, and then restores green. The leaf color mutant only appears white at the seedling stage, and does not continuously show the trait in the whole growth period, and has little influence on maize growth and yield. The InDel molecular marker can be applied to hybrid seed production and purity identification of maize, and can realize rapid and accurate screening of true and false hybrids before sowing or at the seedling stage, and significantly improves hybrid purity.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics, specifically to a molecular marker, detection reagent, and their application that are closely linked to the stage-specific regulatory trait of maize leaf color. Background Technology

[0002] Leaf color mutants, also known as chlorophyll mutants, are caused by mutated genes that lead to abnormal chloroplast structure, affecting chlorophyll synthesis and metabolism, resulting in traits such as whitening, yellowing, and light greening of leaves. These mutants have intuitive and easily identifiable phenotypes, making them ideal morphological markers in maize hybridization breeding. They can be used to quickly distinguish between true and false hybrids, and have significant application value in improving the purity of hybrids. Therefore, identifying genetically stable, phenotypically clear, and non-disruptive superior leaf color genes and applying them to parental genetic improvement is crucial for achieving precise and efficient breeding.

[0003] Currently reported maize leaf color mutants mostly exhibit abnormal leaf color throughout the entire growth period or in the mid-to-late stages of growth. However, resources of stage-specific mutant genes that specifically regulate leaf color only during the seedling stage and can naturally recover green color later remain relatively scarce. These stage-specific leaf color markers only show specific phenotypes during the seedling stage, facilitating early elimination of false hybrids. They can restore normal photosynthesis and growth in the later stages without adversely affecting subsequent plant development, thus possessing higher application potential and safety in practical breeding.

[0004] Meanwhile, to fully leverage the breeding value of superior leaf color marker genes and achieve precise selection and efficient identification of target traits, developing molecular markers closely linked to them has become an essential technical step. Insertion / deletion (InDel) markers are commonly used molecular markers developed based on genomic nucleotide fragment insertion / deletion polymorphisms. They possess advantages such as co-dominance, low cost, good reproducibility, high throughput, and ease of operation, and are widely used in maize genetic analysis, gene mapping, and large-scale assisted breeding, serving as core tools for improving breeding accuracy and selection efficiency. However, to date, there are no reports on InDel molecular markers closely linked to the stage-specific regulatory traits of leaf color in maize seedlings. This lack of efficient molecular tools supports the precise identification of these superior leaf color marker genes, parental introduction verification, and rapid early screening of false hybrids, hindering their large-scale application in hybrid purity identification and restricting further improvements in maize breeding efficiency and seed quality. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a molecular marker, detection reagent and its application that are closely linked to the stage-specific regulatory trait of maize leaf color.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a molecular marker closely linked to the stage-specific regulatory trait of maize leaf color, the molecular marker being named InDel-DF8, located in the 173220444-173220647 bp region on chromosome 6 of maize, with the reference genome being B73 RefGen_v5.

[0007] In maize materials exhibiting stage-specific regulation of leaf color during the seedling stage, the amplified fragment of the molecular marker InDel-DF8 is 163 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1; specifically as follows: ATGTAGCTCGTGTCGTGTGAAGGTTGAACTCGAGACGGTGACAGAAAGAAACTCAGCTGCGCTCGTACAAAGGGAATTCAGCTGCTTCGACGGCACCAAACGATGCCTCAGACAGAGCTCAATGAAGCGCCGCACTGCATCAAAAACTGCAACTCGGTGACAG.

[0008] In normal leaf color allele materials, the amplified fragment of the molecular marker InDel-DF8 is 204 bp in length, and its nucleotide sequence is shown in SEQ ID NO.2; specifically as follows: ATGTAGCTCGTGTCGTGTGAAGGTTGAACTCGAGACGGTGACAGAAAGAAACTCAGCTGCGCTCGTACAAAGGGAATTCAGCTGCTTCGACGGCACCAAACGATGCCTCAGACAGAGCATTCAAAATGGAAGGCGCTATCAGTCCAGCTTTATTACCCCACCATCGAACGCCGCACTGCATCAAAAACTGCAACTCGGTGACAG.

[0009] The stage-specific regulation of maize leaf color during the seedling stage is as follows: the color phenotype and chlorophyll content of maize leaves are developmentally stage dependent; before stage V5, the chlorophyll content decreases, and the leaf color phenotype shows white stripes and gradually turns white; as the development progresses, the white phenotype gradually recovers to the normal leaf color (the white phenotype first gradually turns into light green, and by stage V11 the leaves are completely restored to green).

[0010] Therefore, the maize material of the present invention, which has the characteristic of stage-specific regulation of leaf color in maize seedlings, only exhibits a unique albino phenotype in the seedling stage, which facilitates the early elimination of false hybrids. It can restore normal photosynthesis and growth in the later stage and will not have an adverse effect on the subsequent development of the plant. It has higher application potential and safety in actual breeding.

[0011] The molecular marker InDel-DF8 of this invention is closely linked to the stage-specific regulation of leaf color in maize seedlings. Using the molecular marker InDel-DF8 of this invention, mutants with stage-specific regulation of leaf color in maize seedlings can be identified, which is of great significance for maize breeding and germplasm resource screening.

[0012] In a second aspect, the present invention provides the application of the above-mentioned InDel molecular marker in screening or identifying maize materials with seedling-stage leaf color regulation traits.

[0013] In the above application, the seedling leaf color stage regulation trait is as follows: the color phenotype and chlorophyll content of maize leaves show developmental stage dependence; before the V5 stage, the chlorophyll content decreases, and the leaf color phenotype shows white stripes and gradually turns white; as the development progresses, the white phenotype gradually recovers to normal leaf color.

[0014] A third aspect of the present invention provides a detection reagent comprising: a primer pair for amplifying the aforementioned molecular marker, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; specifically as follows: Upstream primer: 5'-ATGTAGCTCGTGTCGTGTGA-3'; (SEQ ID NO.3) Downstream primer: 5'-CTGTCACCGAGTTGCAGTTT-3'. (SEQ ID NO.4) Furthermore, the detection reagent also includes: DNA template, Taq Master Mix and ddH2O.

[0015] A fourth aspect of the present invention provides the use of the above-described detection reagent in the following (1) or (2): (1) Screening or identifying maize materials with stage-specific regulation traits of leaf color during the seedling stage; (2) Marker-assisted breeding of maize.

[0016] In the above applications, the maize molecular marker-assisted breeding refers to: germplasm innovation of maize leaf color genes or hybrid breeding.

[0017] In the above applications, the method for screening or identifying maize materials with stage-specific regulatory traits of leaf color during the seedling stage includes the following steps: Using the genomic DNA of the maize sample as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO.3 and SEQ ID NO.4, and the amplification products were analyzed by sequencing or gel electrophoresis. If the sequence of the amplified product is as shown in SEQ ID NO.1, or the band in the gel electrophoresis is 163 bp, it is determined to be a maize leaf color mutant with a stage-specific regulatory trait of leaf color.

[0018] If the sequence of the amplified product is as shown in SEQ ID NO.2, or the band in the gel electrophoresis is 204 bp, then it is determined to be wild type.

[0019] If two bands of 204 bp and 163 bp appear simultaneously on gel electrophoresis, it is determined to be a heterozygous genotype.

[0020] Preferably, the PCR amplification reaction system is as follows: 0.5 μL of the upstream primer shown in SEQ ID NO.3 at a concentration of 10 μM; 0.5 μL of the downstream primer shown in SEQ ID NO.4 at a concentration of 10 μM; 1 μL of genomic DNA at a concentration of 100 ng / μL; 5 μL of 2×Taq Master Mix; and 3 μL of ddH2O.

[0021] The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min; and storage at 10℃.

[0022] The beneficial effects of this invention are: This invention is the first to identify an InDel molecular marker on maize chromosome 6 that is closely linked to the stage-specific regulation of leaf color in maize seedlings. This marker allows for precise genotyping of maize populations in the early stages of breeding, efficiently screening for individuals carrying the leaf color regulating gene. These materials gradually develop an albino phenotype before stage V5, subsequently reverting to a naturally green color. The leaf color mutant involved in this invention exhibits albinoness only during the seedling stage and does not continuously express this trait throughout the entire growth period, thus having minimal impact on maize growth and yield. Applying this InDel molecular marker to maize hybrid seed production and purity identification enables rapid and precise screening of true and false hybrids before sowing or during the seedling stage, significantly improving hybrid purity. Attached Figure Description

[0023] Figure 1 Wild-type corn and gral1 Leaf color phenotypes of mutants at stages V1, V2, V4, V5, V7, and V11.

[0024] Figure 2 The molecular marker InDel-DF8 of this invention is effective against B73 and gral1 Alignment results of mutant amplification product sequences.

[0025] Figure 3The 8% PAGE electrophoresis pattern of the PCR amplification product of the molecular marker InDel-DF8 in the F2 population of this invention; wherein, L is the amplification band pattern of the normal leaf color allele material, B is the amplification band pattern of the stage-regulated leaf color trait of maize seedlings, H is the amplification band pattern of the heterozygous genotype, and M is the marker.

[0026] Figure 4 The results of chlorophyll content determination in the F2 population, identified by the molecular marker InDel-DF8 as individuals with stage-specific regulation of maize seedling leaf color (B) and individuals with normal leaf color alleles (L); in the figure, (a) is the result of chlorophyll a content determination; (b) is the result of chlorophyll b content determination; (c) is the result of total chlorophyll content determination. Detailed Implementation

[0027] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0028] Terminology Explanation: V1, V2, V4, V5, V7, and V11 refer to the 1-leaf, 2-leaf, 4-leaf, 5-leaf, 7-leaf, and 11-leaf stages of maize, respectively.

[0029] As mentioned earlier, the study of leaf color mutants helps to elucidate the process of chloroplast biogenesis and is also an important genetic resource. However, existing research still has limitations: most leaf color mutants exhibit persistent abnormal phenotypes, such as chlorosis leading to death throughout the entire growth period, as they cannot effectively perform photosynthesis and are unlikely to survive to maturity, thus limiting in-depth analysis of their mechanism of action throughout the growth period and their application in breeding; in addition, some mutants exhibit long-term yellowing, with leaves remaining yellow throughout the entire growth period, resulting in low light energy utilization efficiency and hindering crop yield improvement.

[0030] The inventor discovered a corn leaf color mutant in the field. gral1 Its leaf color phenotype exhibits a phased change: before stage V5, the leaves gradually turn white, then gradually return to green, and finally turn completely green in stage V11. The inventors... gral1 Using maize inbred line B73 as the female parent and maize as the male parent, an F2 segregating population was constructed to finely map genes regulating leaf color at the seedling stage. The constructed F2 segregating population was planted in the field, and leaf color phenotypic identification was performed. Total DNA was extracted from leaves of albino plants, and existing SSR markers from the synthesized maize IBM map and self-developed SSR markers were used to further analyze the genes involved. gral1 Polymorphic screening was performed on mutants and parents. Primers were used to encrypt the genome based on the published maize genome, ultimately locating the target gene at [location missing]. InDel-DF8 and InDel-859 The physical distance between the two molecular markers is 83.7 kb. Sequencing within the localization region revealed that, compared to the wild type, gral1 The mutant has an insertion of 9 nucleotides and a deletion of 50 nucleotides. This region can be used as an InDel molecular marker closely linked to the stage-specific regulatory trait of leaf color in maize seedlings, and is named InDel-DF8.

[0031] Based on this InDel molecular marker, the present invention further designed specific primers for specifically amplifying the above-mentioned InDel molecular marker, as follows: Upstream primer: 5'-ATGTAGCTCGTGTCGTGTGA-3' (SEQ ID NO.3) Downstream primer: 5'-CTGTCACCGAGTTGCAGTTT-3' (SEQ ID NO.4) BLAST analysis of the above-mentioned specific primers, based on the existing maize genome sequence, revealed that the molecular marker InDel-DF8 is located at 173220444-173220647 bp on maize chromosome 6, with the physical location referenced from the B73RefGen_v5 genome version.

[0032] The molecular marker InDel-DF8 of this invention can accurately identify maize leaf color traits at an early stage, which is beneficial for identifying false hybrids. This invention was proposed based on this.

[0033] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0034] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions.

[0035] Example 1: Obtaining InDel molecular markers closely linked to the stage-specific regulatory trait of leaf color in maize seedlings 1. Obtaining mutants that regulate leaf color at different stages in maize seedlings The inventors identified a mutant exhibiting stage-specific leaf color changes during the seedling stage within the genetic context of the maize inbred line J220 (wild-type, WT), and named it... gral1 .

[0036] Compared to the J220, gral1 During the seedling stage, the leaves gradually lighten and exhibit an chlorotic phenotype, subsequently recovering their green color as the plant develops, until the leaves are almost or completely green again by stage V11. Chlorophyll content measurements indicate that during the leaf whitening stage... gral1 The chlorophyll content of the chlorophyll-rich variety was significantly lower than that of the wild type, but after revegetation, the difference between the chlorophyll-rich variety and the wild type was not significant.

[0037] 2. Genetic analysis use gral1 The mutant was crossed with the normal inbred line "Huangzao Si". The F1 generation showed normal phenotype development. In the F2 segregating population, the ratio of wild-type phenotype plants to mutant phenotype plants was determined by chi-square test. The results showed that χ²=0.625, df=1, χ²0.05,1=3.841, and P>0.05, which met the 3:1 segregation ratio. This indicates that the leaf color stage regulation trait is controlled by a pair of recessive nuclear genes.

[0038] Table 1: Genetic Analysis 3. Location of genes regulating leaf color during the seedling stage in maize pass gral1 The F1 generation was obtained by crossing with a normal green-leaved plant (Huangzao 4). After self-pollination of the F1 generation, the F2 generation was obtained. Using white plants in the F2 generation, genetic exchange was conducted to determine the target gene locus, which was ultimately located. Genetic analysis showed that... gral1 The leaf albino phenotype in the offspring of hybridization with 'Huangzao 4' is controlled by a single recessive nuclear gene. Preliminary findings suggest... ZmGRAL1 Located on the long arm of maize chromosome 6 p-umc2170 and p-umc2059 Markings.

[0039] To further narrow the location range, we expanded the group, including the markers. p-umc2170 and p-umc2059 Develop SSR tags between tags, utilizing ( gral1 (Huang Zao Si) Of the 2112 mutant phenotype individuals in the F2 population, the target interval was further narrowed down to M1 to M2 The physical distance between the two markers is 4.7 Mb. Because in M1 to M2 Since there were no available markers and the recombinant plants were no longer being exchanged, we grouped them. gral1 A new combination with the B73, and in M1 to M2 The design incorporates 12 pairs of SSR markers, utilizing ( gral1 The 4235 mutant phenotype individuals in F2 of / B73) narrowed the target region to M11 and M12 Between the two markers, which are 83.7 Kb apart.

[0040] Table 2: SSR Molecular Markers Sequencing revealed that, compared to the wild type, gral1The mutant contains an insertion of 9 nucleotides and a deletion of 50 nucleotides. Figure 2 This region can serve as an InDel molecular marker closely linked to the stage-specific regulatory trait of leaf color in maize seedlings, named InDel-DF8, located in the 173220444-173220647 bp region on chromosome 6 of maize, with the reference genome being B73RefGen_v5; in maize materials with stage-specific regulatory traits of leaf color in maize seedlings, the amplified fragment of the InDel molecular marker is 163 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1; in normal leaf color allele materials, the amplified fragment of the InDel molecular marker is 204 bp in length, and its nucleotide sequence is shown in SEQ ID NO.2.

[0041] Example 2: Application and verification of InDel molecular markers closely linked to the stage-specific regulation of leaf color in maize seedlings. Based on the molecular marker InDel-DF8 screened in Example 1, primer pairs for amplifying the molecular marker were designed. The designed primer pair sequences are as follows: Upstream primer: 5'-ATGTAGCTCGTGTCGTGTGA-3' (SEQ ID NO.3) Downstream primer: 5'-CTGTCACCGAGTTGCAGTTT-3' (SEQ ID NO.4) Using maize inbred line B73 and gral1 Using mutants as parents, new F2 segregating populations were constructed. Using the molecular marker InDel-DF8 and designed primer pairs, the stage-specific regulatory traits of maize seedling leaf color in the F2 segregating populations were detected, as detailed below: Using the genomic DNA of the maize sample as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO.3 and SEQ ID NO.4, and the size of the amplification product was used to determine the amplification result.

[0042] If the molecular weight of the PCR amplification product of the maize sample to be tested is 163 bp, the sample contains alleles that regulate the leaf color stage of maize seedlings; if the molecular weight of the PCR amplification product of the maize sample to be tested is 204 bp, the sample contains alleles that indicate normal green leaves.

[0043] The PCR amplification reaction system consisted of: 0.5 μL of the 10 μM upstream primer shown in SEQ ID NO.3; 0.5 μL of the 10 μM downstream primer shown in SEQ ID NO.4; 1 μL of genomic DNA at a concentration of 100 ng / μL; 5 μL of 2×Taq Master Mix; and 3 μL of ddH2O.

[0044] The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min; storage at 10℃. The amplified fragments described above were subjected to electrophoresis on an 8% polyacrylamide gel (e.g.) Figure 3 As shown in the figure, if a band of 163 bp is amplified, it is identified as maize material with the stage-specific regulation of leaf color in maize seedlings (marked as B); if a band of 204 bp is amplified, it is identified as normal leaf color allele material (marked as L); if two bands of 204 bp and 163 bp are amplified, it indicates that the maize is heterozygous (marked as H).

[0045] The chlorophyll content in the leaves of maize materials identified using the molecular marker InDel-DF8 as exhibiting stage-specific leaf color changes during the seedling stage and normal green leaf color was determined using the following method: Leaves from maize materials (labeled B) and normal leaf color alleles (labeled L) with similar growth at three stages—before albinoing (V1), albinoing state (V4), and after regreening (V11)—were identified using InDel-DF8. Pigments were extracted from the fresh leaf tissues using 95% ethanol. The OD values ​​of chlorophyll a and chlorophyll b at wavelengths of 665 nm and 649 nm were measured using a spectrophotometer TU-1950. The concentrations of chlorophyll a (Ca), chlorophyll b (Cb), and total chlorophyll (Chl) were calculated using the Lichtenthaler revised formula. Finally, the content of each photosynthetic pigment was calculated using the formula: Chlorophyll content (mg / g) = [pigment concentration × extraction liquid volume × dilution factor] / sample fresh weight.

[0046] (1) Ca (mg / l) = 13.95 × OD 665 -6.88×OD 649 (2) Cb (mg / l) = 24.96 × OD 649 -7.32×OD 665 (3) Chl = Ca + Cb The results are as follows Figure 4 As shown, chlorophyll content analysis indicated that material B was not significantly different from material L at stages V1 and V11, but was significantly lower than material L at stage V4, suggesting that this leaf color mutation phenotype mainly occurred at a specific seedling stage and could return to normal later.

[0047] The above results demonstrate that the molecular marker InDel-DF8 of this invention can be used to identify the stage-specific regulatory traits of leaf color in maize seedlings, and the results are accurate and reliable.

[0048] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A molecular marker closely linked to the stage-specific regulatory trait of maize leaf color, characterized in that, The molecular marker, named InDel-DF8, is located in the 173220444-173220647 bp region on chromosome 6 of maize, with the reference genome being B73RefGen_v5. In maize materials exhibiting stage-regulated leaf color traits, the amplified fragment of InDel-DF8 is 163 bp in length, and its nucleotide sequence is shown in SEQ ID NO.

1. In maize materials exhibiting normal leaf color traits, the amplified fragment of InDel-DF8 is 204 bp in length, and its nucleotide sequence is shown in SEQ ID NO.

2.

2. The application of the detection reagent in screening or identifying maize materials with seedling-stage regulation of leaf color traits, characterized in that, The detection reagent comprises: a primer pair for amplifying the molecular marker of claim 1, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; The method for screening or identifying maize materials that exhibit leaf color traits that are regulated at the seedling stage includes the following steps: Using the genomic DNA of the maize sample as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO.3 and SEQ ID NO.4, and the amplification products were analyzed by sequencing or gel electrophoresis. If the amplification product is a single 163bp band, the maize to be tested is determined to have a seedling stage-regulated leaf color trait; if the amplification product is a single 204bp band, the maize to be tested is determined to be a homozygous type of normal leaf color allele.

3. The application according to claim 2, characterized in that, The detection reagent also includes: DNA template, TaqMaster Mix and ddH2O.

4. The application according to claim 2, characterized in that, The PCR amplification reaction system consisted of 0.5 μL of the upstream primer shown in SEQ ID NO.3 at a concentration of 10 μM; 0.5 μL of the downstream primer shown in SEQ ID NO.4 at a concentration of 10 μM; 1 μL of genomic DNA at a concentration of 100 ng / μL; 5 μL of 2×Taq Master Mix; 3 μL of ddH2O; The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min; and storage at 10℃.

Images