A ZmCPS5 gene related to chlorophyll content in corn leaves and its SNP molecular marker and application

Abstract

The present invention discloses a ZmCPS5 gene related to the chlorophyll content of corn leaves and its SNP molecular marker and application, which belong to the field of plant genetic engineering technology. The present invention provides a ZmCPS5 gene related to the chlorophyll content of corn leaves, and the nucleotide sequence of the ZmCPS5 gene is shown in SEQ ID NO: 1. The present invention also provides a ZmCPS5 gene SNP molecular marker related to the chlorophyll content of corn leaves, the SNP is located at the 10443485 site of chromosome 8 in the 3′-UTR region of the ZmCPS5 gene, and the polymorphism is G / A. The gene ZmCPS5 provided by the present invention provides a reliable target for the application of high-light-efficiency genetic engineering breeding of corn, and the provided SNP molecular marker provides a reliable molecular marker for the application of high-light-efficiency molecular marker-assisted selection breeding of corn.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a ZmCPS5 gene related to the chlorophyll content of corn leaves, a SNP molecular marker thereof, and applications thereof. Background Art

[0002] Increasing corn yield by leveraging hybrid vigor is a traditional breeding method, but it has drawbacks such as high blindness and a significant workload. With the rapid development of corn molecular biology, genetic engineering combined with marker-assisted selection has become a new approach for rapidly breeding varieties with targeted traits.

[0003] Photosynthesis is a crucial process for plants to convert light energy into organic matter. Chlorophyll, the primary photosynthetic pigment, facilitates electron transfer within the chloroplasts of higher plants. Increased chlorophyll content significantly enhances a plant's ability to absorb light, thereby increasing photosynthetic efficiency. While genes associated with chlorophyll content have been reported in maize, the genetic segments (locus) regulating chlorophyll content remain largely undetermined.

[0004] Linkage analysis is a genetic research method that uses linkage relationships between phenotype and genotype to identify quantitative trait loci (QTLs) controlling target traits and subsequently identify potential candidate genes. In recent years, the rapid development of high-throughput sequencing technologies has enabled the identification of large-scale molecular markers across the genome, and linkage analysis has become widely used in animal and plant research. Therefore, using linkage analysis to elucidate the genetic basis of chlorophyll content in maize leaves could provide important molecular markers and gene targets for improving maize yield-related traits. Summary of the Invention

[0005] The present invention aims to provide a ZmCPS5 gene and its SNP molecular marker related to the chlorophyll content of maize leaves and its application to solve the problems existing in the above-mentioned prior art. The present invention identifies the gene ZmCPS5 and its SNP molecular marker that can control the chlorophyll content of maize leaves.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: A ZmCPS5 gene related to the chlorophyll content of corn leaves, the nucleotide sequence of the ZmCPS5 gene is shown in SEQ ID NO: 1.

[0008] Technical solution 2: Application of the ZmCPS5 gene or the protein encoded thereby in regulating the chlorophyll content in corn leaves, wherein the amino acid sequence of the protein encoded thereby is shown in SEQ ID NO: 2.

[0009] Technical solution three: Application of a recombinant vector containing the ZmCPS5 gene in regulating the chlorophyll content of corn leaves.

[0010] Technical solution 4: Application of the host bacteria containing the recombinant vector in regulating the chlorophyll content of corn leaves.

[0011] Technical Solution 5: A SNP molecular marker of the ZmCPS5 gene related to the chlorophyll content of corn leaves, the SNP is located at position 10443485 of chromosome 8 in the 3′-UTR region of the ZmCPS5 gene, and the polymorphism is G / A; the nucleotide sequence of the ZmCPS5 gene is shown in SEQ ID NO: 1.

[0012] Technical Solution 6: Application of a detection reagent for detecting the SNP molecular marker of the ZmCPS5 gene in detecting the chlorophyll content of corn leaves.

[0013] Technical Solution 7: A primer set for amplifying the SNP molecular marker of the ZmCPS5 gene, comprising primers with sequences shown as SEQ ID NO: 22 and SEQ ID NO: 23.

[0014] Technical Solution 8: An application of the primer set in preparing a kit for identifying the chlorophyll content in corn leaves.

[0015] Technical Solution Nine: Application of the ZmCPS5 gene SNP molecular marker in molecular marker-assisted breeding related to chlorophyll content in maize leaves.

[0016] Furthermore, it includes the application of the allelic variation of the ZmCPS5 gene SNP molecular marker in the breeding of high light efficiency corn.

[0017] The present invention discloses the following technical effects:

[0018] Through linkage analysis, the present invention identified the regulatory gene ZmCPS5, which controls chlorophyll content in maize leaves. By amplifying the gene's promoter and gene body and employing candidate gene association analysis, a single-nucleotide polymorphism (SNP) marker, SNP-8-10443485, was identified in its 3′-UTR region, which is significantly associated with chlorophyll content. This gene provides a reliable target for genetic engineering breeding of maize with high light efficiency. Validation results from the present invention demonstrate that this marker can effectively distinguish leaf chlorophyll content, providing a reliable molecular marker for use in marker-assisted selection breeding of maize with high light efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 are the phenotypic values of the inbred lines in the IBM Syn10 DH population under three environments; A represents the phenotypic values of the population parents Mo17 and B73 under three environments; B represents the phenotypic values of the 230 inbred lines under three environments; CCFSS represents the chlorophyll content of the fifth leaf at the seedling stage; CCEFS represents the chlorophyll content of the ear leaf at the filling stage; YA, CZ, and YN represent Ya'an, Sichuan, Chongzhou, Sichuan, and Jinghong, Yunnan, respectively; *** and ** represent significance at the P < 0.001 and P < 0.01 levels, respectively;

[0021] Figure 2 is the QTL mapping result of chlorophyll content in the fifth leaf at the seedling stage; where CCFSS represents the chlorophyll content in the fifth leaf at the seedling stage;

[0022] Figure 3 is the QTL mapping result of chlorophyll content in ear leaves during the grain filling period; where CCEFS represents the chlorophyll content in ear leaves during the grain filling period;

[0023] Figure 4 is the result of association analysis based on the ZmCPS5 gene; CCFSS represents the chlorophyll content of the fifth leaf at the seedling stage; CCEFS represents the chlorophyll content of the ear leaf at the filling stage;

[0024] Figure 5 The following are the statistical analysis results of the phenotypic values of the two haplotypes divided based on the molecular marker SNP-8-10443485 in the natural population; A is the statistical analysis result of the chlorophyll content of the fifth leaf at the seedling stage for different haplotypes; B is the statistical analysis result of the chlorophyll content of the ear leaf at the grain filling stage for different haplotypes; ** and * represent significant differences at the P < 0.01 and P < 0.05 levels, respectively.

[0025] Figure 6 The validation results of the molecular marker SNP-8-10443485 in the segregating population; A is the statistical analysis result of the chlorophyll content in the fifth leaf at the seedling stage based on the different allele variations (G / A) amplified by SNP-8-10443485; B is the statistical analysis result of the chlorophyll content in the ear leaf at the filling stage based on the different allele variations (G / A) amplified by SNP-8-10443485; *** represents significance at the P < 0.001 level. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1

[0032] 1. Phenotypic identification of population materials

[0033] A population of 230 recombinant inbred lines (RIs) from IBM Syn10 DH was planted at experimental bases in Chongzhou, Sichuan Province (2021, 30.30°N, 103.07°E), Ya'an, Sichuan Province (2021, 29.59°N, 102.57°E), and Jinghong, Yunnan Province (2022, 22.00°N, 100.79°E). A randomized block design was used, with three biological replicates for each inbred line. Chlorophyll content was measured in the fifth leaf at the seedling stage (30 days after sowing) and in the ear-position leaves at the grain-filling stage (5 days after pollination) using a SPAD 502Plus chlorophyll meter.

[0034] The chlorophyll content of the fifth leaf of the seedling stage and the chlorophyll content of the ear leaf of the parent B73 of the IBM Syn10 DH population were significantly higher than those of Mo17 ( Figure 1 A), the chlorophyll content of the fifth leaf at the seedling stage was the highest in the Ya'an environment, and the chlorophyll content of the ear leaf at the filling stage was the highest in the Chongzhou environment ( Figure 1 B).

[0035] 2. Linkage analysis to identify QTLs and candidate genes associated with leaf chlorophyll content

[0036] Best linear unbiased predictions (BLUPs) were performed based on the phenotypic values from the three environments, and the BLUP values were used as the final phenotypic values for subsequent analysis. The population genotype was obtained from a previous study and included 6,618 bin markers. Linkage analysis was performed using WinQTLCart 2.5 software using composite interval mapping, combining BLUP values and bin markers. The QTL detection threshold was set at LOD = 2.5. A single QTL (qCCFSS8-1) was identified that controls chlorophyll content in the fifth leaf at the seedling stage ( Figure 2 ), 6 QTLs controlling the chlorophyll content of ear leaves during the grain filling period (qCCEFS4-1, qCCEFS7-1, qCCEFS8-1, qCCEFS9-1, qCCEFS9-2, qCCEFS10-1) ( Figure 3 ). The 7 QTLs were distributed on chromosomes 4, 7, 8, 9, and 10. The LOD values ranged from 2.55 to 4.18 ( Figure 2 and Figure 3 ), and the explained phenotypic variation rate ranged from 3.65% to 6.55%. By searching the MaizeGDB database, 7 QTLs contained a total of 219 candidate genes. Among them, the lead QTL (qCCFSS8-1) contained 16 candidate genes, and gene annotation showed that Zm00001d008497 (ZmCPS5) encodes chlorophyll synthesis protein (chloroplast proteinsynthesis5), which is closely related to chlorophyll content. Therefore, ZmCPS5 was identified as a candidate gene regulating the chlorophyll content of maize leaves. The amino acid sequence of its encoded protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the ZmCPS5 gene is shown in SEQ ID NO: 1.

[0037] SEQ ID NO: 1:

[0038]

[0039] MALALQWPLQFPLQLQARPPAVTAGHHRRRHRVLAVCRSPPLPARCCASAAAAADTGKAQTAARRAYPFDEIEPRWQRHWEEHRTFRTLDIGEGLDTSKPKCYILDMFPYPSGA GLHVGHPLGYTATDILSRFKRMKGFNVLHPMGWDAFGLPAEQYAIQTGTHPKITTERNIERFRTQLKSLGFSYDWDREISTTEPGYYKWTQWIFLQLLKRGLAYQAGIDILQSG.

[0040] 3. Association analysis based on the ZmCPS5 gene

[0041] To identify the variation within gene ZmCPS5 that is significantly correlated with chlorophyll content, PCR was used to amplify its promoter (the promoter is 2000 bp, the nucleotide sequence is shown in SEQ ID NO:3; the primer sequences used to amplify the promoter are shown in SEQ ID NO:4-SEQ ID NO:7) and gene body nucleotide sequence (the gene body nucleotide sequence is shown in SEQ ID NO:1; the amplification primer sequences used to amplify the gene ZmCPS5 body are shown in SEQ ID NO:8-SEQ ID NO:21) in a natural population containing 300 inbred lines.

[0042] SEQ ID NO:3:

[0043]

[0044] ①F:CGTAGCAGAGCCGTTAAGGT(SEQ ID NO:4);R:AGACGAGAAAGGCTAAGGCG(SEQ IDNO:5);

[0045] ②F:CCTCCCTCCTCTTACACCCA(SEQ ID NO:6);R:GTTCCGGTAAGGCCCTTTGA(SEQ IDNO:7);

[0046] ③F:CCCTTGGATACACGGCAACT(SEQ ID NO:8);R:ACACAAAGAAGCACCCCACA(SEQ IDNO:9);

[0047] ④F:TGGCCCGAGAGTATCAAGGA(SEQ ID NO:10);R:GTGTCAGGCCTCGTTGTGTA(SEQ IDNO:11);

[0048] ⑤F:TTGGGGTGAACCTTTCCCTG(SEQ ID NO:12);R:TCCCCAGTACCAGTTGGAGT(SEQ IDNO:13);

[0049] ⑥F:GACCCTTCATCTGGGAAGCC(SEQ ID NO:14);R:CACCATGTAGCACACCCCTT(SEQ IDNO:15);

[0050] ⑦F:GAGCCCTTCAAGTGCCTGAT(SEQ ID NO:16);R:CTCCACGTTCGTCCAAGGAA(SEQ IDNO:17);

[0051] ⑧F:ACAAGCCTTTCCCCCAAACA(SEQ ID NO:18);R:TCACCCAACCCAGTTTACGG(SEQ IDNO:19);

[0052] ⑨F:TTTGGTTTCGCCTCGGACAT(SEQ ID NO:20);R:CCTCAGAGCACGCTTTGTCA(SEQ IDNO:21)。

[0053] The PCR amplification system was as follows: 2× buffer 12.5 μL, ddH2O 8.5 μL, Primer F 1 μL, Primer R 1 μL, cDNA 1 μL, dNTPs 0.5 μL, and enzyme 0.5 μL. The PCR amplification program was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 25 s, 34 cycles, 72°C extension for 5 min, and storage at 12°C for 10 min.

[0054] The amplified sequences were aligned to the B73v4 reference genome, and a total of 38 SNP variations were obtained, of which 20 SNPs were located in the 3′-UTR region, 10 SNPs were located in the intron region, 2 SNPs were located in the promoter region, and 6 SNPs were located in the exon region (3 synonymous mutations and 3 missense mutations). Combining the phenotypic values (chlorophyll content of the fifth leaf at the seedling stage and chlorophyll content of the ear leaf at the filling stage) and genotypes (38 SNPs) of 319 inbred lines, the FarmCPU model was used for association analysis. A total of 6 SNPs were detected to be significantly associated with the chlorophyll content of the fifth leaf at the seedling stage (P<0.05); 1 SNP was significantly associated with the chlorophyll content of the ear leaf at the filling stage (P<0.05) ( Figure 4 Among them, marker SNP-8-10443485 (G / A) located in the 3′-UTR region was most significantly associated with the chlorophyll content of the fifth leaf at the seedling stage (lead SNP).

[0055] 4. Application of molecular marker SNP-8-10443485

[0056] Based on marker SNP-8-10443485, the natural population was divided into two haplotypes (Hap I:G; Hap II:A). Among them, Hap I included 48 inbred lines and Hap II included 252 inbred lines. The chlorophyll content of the fifth leaf at the seedling stage and the chlorophyll content of the ear leaf at the filling stage in Hap I (G) were significantly higher than those in Hap II (A) (P<0.05) ( Figure 5 Therefore, Hap I (G) is the superior haplotype, and Hap II (A) is the non-superior haplotype.

[0057] To further verify the marker, it was PCR amplified in the IBM Syn 10DH population, and the primer sequences for PCR amplification were shown in SEQ ID NO: 22 and SEQ ID NO: 23 (F: ACTCGCAACACCAGAAAGGA (SEQ ID NO: 22); R: ACAAGCACCATGCCACAATG (SEQ ID NO: 23)).

[0058] The PCR amplification system was as follows: 2× buffer 12.5 μL, ddH2O 8.5 μL, Primer F 1 μL, Primer R 1 μL, cDNA 1 μL, dNTPs 0.5 μL, and enzyme 0.5 μL.

[0059] The PCR amplification procedure was as follows: ① pre-denaturation at 95°C for 3 min; ② denaturation at 95°C for 15 s; ③ annealing at 58°C for 15 s; ④ extension at 72°C for 30 s; ②-④ were set for 35 cycles; ⑤ incubation at 72°C for 5 min, and holding at 12°C.

[0060] The amplified products were sequenced to detect the allelic variation of the marker position. The results showed that 37 materials had a G allelic variation at the marker position, and 193 materials had an A allelic variation at the marker position. The two types of allelic variation showed significant differences in the chlorophyll content of the fifth leaf at the seedling stage and the chlorophyll content of the ear leaf at the filling stage (P<0.05) ( Figure 6 A and B). It further shows that marker SNP-8-10443485 (G / A) can effectively identify maize materials with high chlorophyll content.

[0061] This study identified the gene ZmCPS5, which controls chlorophyll content in maize leaves, and a molecular marker SNP-8-10443485, significantly associated with leaf chlorophyll content, within the 3′-UTR region of this gene. The G allele of this marker was confirmed to be a superior allele. Furthermore, amplification of this marker in a population confirmed its ability to effectively differentiate leaf chlorophyll content among different maize inbred lines. This invention provides a target gene for breeding maize varieties with high light efficiency and an effective molecular marker for selecting maize materials with high light efficiency.

[0062] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for detecting a ZmCPS5 gene SNP molecular marker in detecting chlorophyll content in corn leaves, characterized in that: The SNP is located at position 10443485 of chromosome 8 in the 3′-UTR region of the ZmCPS5 gene, and the polymorphism is G / A; the nucleotide sequence of the ZmCPS5 gene is shown in SEQ ID NO: 1; and the reference genome of the 10443485 position is B73v4.

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