ZmCPS5 gene related to chlorophyll content of corn leaves as well as SNP (Single Nucleotide Polymorphism) molecular marker and application of ZmCPS5 gene

Abstract

The invention discloses a ZmCPS5 gene related to the chlorophyll content of corn leaves as well as an SNP (Single Nucleotide Polymorphism) molecular marker and application thereof, and belongs to the technical field of plant genetic engineering. The invention provides a ZmCPS5 gene related to the chlorophyll content of corn leaves. The nucleotide sequence of the ZmCPS5 gene is as shown in SEQ ID NO: 1. The invention further provides an SNP molecular marker of the ZmCPS5 gene related to the chlorophyll content of the corn leaves, the SNP molecular marker is located at the 10443485 site of the No.8 chromosome of the 3 '-UTR region of the ZmCPS5 gene, and the polymorphism is G / A. The gene ZmCPS5 provided by the invention provides a reliable target for corn high-photosynthetic-efficiency gene engineering breeding application, and the provided SNP molecular marker provides a reliable molecular marker for corn high-photosynthetic-efficiency molecular marker-assisted selective breeding application.

Description

Technical Field

[0001] The 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 and a SNP molecular marker and application thereof. Background Art

[0002] Increasing corn yield by utilizing hybrid vigor is the main means of traditional breeding, but it has disadvantages such as strong blindness and huge workload. With the rapid development of corn molecular biology, the use of genetic engineering combined with molecular marker-assisted selection has become a new method for rapid breeding of target trait varieties.

[0003] Photosynthesis is an important way for plants to convert light energy into organic matter. Chlorophyll is the main photosynthetic pigment, which can promote electron transfer in chloroplasts of higher plants. The increase in chlorophyll content can significantly improve the plant's ability to absorb light, thereby improving the efficiency of photosynthesis. In corn, although there are reports of genes related to chlorophyll content, the genetic segments (locus) that regulate it have not yet been fully deciphered.

[0004] Linkage analysis is a genetic research method that identifies quantitative trait loci (QTLs) that control target traits based on the linkage relationship between phenotype and genotype, and then explores potential candidate genes. In recent years, based on the rapid development of high-throughput sequencing technology, large-scale molecular markers on the genome have been identified, and linkage analysis has been widely used in animal and plant related research. Therefore, using linkage analysis to analyze the genetic basis of chlorophyll content in maize leaves can provide important molecular markers and important gene targets for the improvement of maize yield-related traits. Summary of the invention

[0005] The purpose of the present invention is to provide a ZmCPS5 gene and its SNP molecular marker related to the chlorophyll content of corn 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 corn 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 by it in regulating the chlorophyll content of corn leaves, the amino acid sequence of the protein encoded by the ZmCPS5 gene 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 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, wherein 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 in SEQ ID NO:22 and SEQ ID NO:23.

[0014] Technical Solution 8: An application of the primer set in the preparation of a kit for identifying the chlorophyll content of 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 SNP molecular marker of the ZmCPS5 gene in the breeding of corn with high light efficiency.

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

[0018] The present invention identified the regulatory gene ZmCPS5 that controls the chlorophyll content of corn leaves through linkage analysis. By amplifying the promoter and gene body of the gene and using candidate gene association analysis, a SNP molecular marker SNP-8-10443485 significantly associated with chlorophyll content was mined in its 3′-UTR region. The gene provides a reliable target for the application of corn high-light-efficiency genetic engineering breeding. The verification results of the present invention show that the marker can effectively distinguish the chlorophyll content of leaves, providing a reliable molecular marker for the application of corn high-light-efficiency molecular marker-assisted selection breeding. 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

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

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

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

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

[0024] Figure 5 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 of different haplotypes; B is the statistical analysis result of the chlorophyll content of the ear leaf at the filling stage of different haplotypes; **, * represent significant 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 segregation population; A is the statistical analysis result of the chlorophyll content in the 5th 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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] The IBM Syn10 DH population containing 230 recombinant inbred lines was planted in the experimental bases of Chongzhou City, Sichuan Province (2021, 30.30°N, 103.07°E), Ya'an City, Sichuan Province (2021, 29.59°N, 102.57°E), and Jinghong City, Yunnan Province (2022, 22.00°N, 100.79°E). The experiment adopted a randomized block design, and three biological replicates were set for each inbred line. The chlorophyll content of the fifth leaf in the seedling stage and the chlorophyll content of the ear leaf in the filling stage were measured using a SPAD 502Plus chlorophyll meter at the seedling stage (30 days after sowing) and the filling stage (5 days after pollination).

[0034] The chlorophyll content of the fifth leaf of the seedling stage and the chlorophyll content of the ear leaf at the grain filling stage in the parent B73 of the IBM Syn10 DH population were significantly higher than those in 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 related to leaf chlorophyll content

[0036] The best linear unbiased prediction (BLUP) was performed based on the phenotypic values ​​of the three environments, and the BLUP values ​​were used as the final phenotypic values ​​for subsequent analysis. The population genotype was obtained from previous studies, including 6,618 bin markers. The WinQTLCart 2.5 software was used to perform linkage analysis based on the composite interval mapping method, combining the BLUP values ​​and bin markers. The QTL detection threshold was set to LOD = 2.5. A total of one QTL (qCCFSS8-1) controlling the chlorophyll content of the fifth leaf at the seedling stage was identified ( Figure 2 ), 6 QTLs controlling the chlorophyll content of ear leaves during the 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 for regulating the chlorophyll content of maize leaves, and 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 ZmCPS5 gene

[0041] In order to clarify the variation within gene ZmCPS5 that is significantly correlated with chlorophyll content, PCR was used to amplify its promoter (the promoter is 2000 bp, and 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 the 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: 2× buffer 12.5μL, ddH2O 8.5μL, Primer F 1μL, Primer R 1μL, cDNA 1μL, dNTPs 0.5μL, enzyme 0.5μL; the PCR amplification program was: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 58℃ annealing for 15s, 72℃ extension for 25s, 34 cycles, 72℃ extension for 5min, and 12℃ storage for 10min.

[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 the 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, the 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 contained 48 inbred lines and Hap II contained 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 a superior haplotype, and Hap II (A) is a 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: 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 procedure of PCR amplification 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 maintained 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] The present invention identified the gene ZmCPS5 that controls the chlorophyll content of corn leaves, and identified the molecular marker SNP-8-10443485 that is significantly associated with the chlorophyll content of leaves in the 3′-UTR region of the gene, confirming that the allele variation G of the marker is an excellent allele variation. In addition, by amplifying the marker in the population, it was confirmed that it can effectively distinguish the chlorophyll content of leaves of different corn inbred lines. The invention provides a target gene for corn high-light-efficiency breeding and an effective molecular marker for the selection of corn high-light-efficiency materials.

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A ZmCPS5 gene related to chlorophyll content in maize leaves, characterized in that: The nucleotide sequence of the ZmCPS5 gene is shown in SEQ ID NO:

1.

2. The use of the ZmCPS5 gene or the protein encoded by it in regulating the chlorophyll content of corn leaves according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the ZmCPS5 gene is shown in SEQ ID NO:

2.

3. Use of a recombinant vector containing the ZmCPS5 gene according to claim 1 in regulating the chlorophyll content of corn leaves.

4. Use of a host bacterium containing the recombinant vector according to claim 3 in regulating the chlorophyll content of corn leaves.

5. A ZmCPS5 gene SNP molecular marker associated with chlorophyll content in maize 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.

6. Use of a detection reagent for detecting the SNP molecular marker of the ZmCPS5 gene according to claim 5 in detecting the chlorophyll content of corn leaves.

7. A primer set for amplifying the SNP molecular marker of the ZmCPS5 gene according to claim 5, characterized in that: Primers comprising sequences shown as SEQ ID NO:22 and SEQ ID NO:

23.

8. Use of the primer set according to claim 7 in preparing a kit for identifying the chlorophyll content of corn leaves.

9. Use of the ZmCPS5 gene SNP molecular marker described in claim 5 in molecular marker-assisted breeding related to chlorophyll content in maize leaves.

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