Maize flowering period regulation gene Zm00001d032922 as well as encoding protein, molecular marker and application of corn flowering period regulation gene Zm00001d032922

Abstract

The invention discloses a corn flowering period regulation gene Zm00001d032922 as well as an encoding protein, a molecular marker and application of the corn flowering period regulation gene Zm00001d032922. According to the application, a key gene Zm00001d032922 which affects differentiation from apical meristem to tassel is cloned by utilizing a reproductive defect natural mutant id1 found in a corn field breeding line selection process, and the gene encodes a C2H2 type zinc finger protein transcription factor; the Zm00001d032922 gene is used for regulating and controlling the flowering period of the corn, and has an important value for improving the growth period of the corn; the identification of the excellent haplotype of the Zm00001d032922 provides technical support and excellent germplasm resources for the utilization of the gene. By utilizing the gene and the molecular marker disclosed by the invention, the creation of high-yield or high-quality breeding materials of corn can be facilitated, the breeding period of new crop varieties can be shortened, the breeding cost can be reduced, and the breeding efficiency can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a gene regulating the flowering period of maize. Zm00001d032922 , its encoded protein, SNP sites, molecular markers and their applications. Background Art

[0002] corn( Zea mays L.) is one of the important food and feed crops. Maize flowering period is one of the important breeding target traits, which is closely related to maize's regional adaptability, yield, resistance and other agronomic traits. Analyzing the genetic basis of maize flowering period has important practical guiding significance for making full use of the diverse germplasm resources of maize and breeding excellent maize varieties with suitable flowering period. In recent years, many scholars have carried out a lot of research work on the regulation of maize flowering period, isolated and cloned many genes that regulate maize flowering, and constructed a preliminary molecular network for regulating maize flowering. However, since the flowering period of maize is an extremely complex biological process, its molecular mechanism still needs to be further analyzed.

[0003] INDETERMINATE1 ( ID1 ) is the only gene found so far that plays a key role in controlling the transition to reproductive growth in maize. id1 The mutant plants were in a prolonged vegetative growth state and showed a delayed transition to flowering, occasionally forming inflorescences with reproductive characteristics, and expression analysis showed that ID1 It is expressed only in immature leaves but not in the apical meristem, indicating ID1 May regulate a long-range, leaf-derived floral signal. ID1 As a plant-specific zinc finger protein transcription factor, the long-distance leaf-derived floral signal it regulates is still unclear.

[0004] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0005] The present invention applies to the use of reproductive conversion natural mutants found in the process of corn field breeding selection id1 Cloning of key genes affecting flowering time Zm00001d032922 , which encodes a C2H2-type zinc finger protein transcription factor. Zm00001d032922 Regulating the flowering period of corn provides new genetic resources for breeding corn varieties with suitable growth periods.

[0006] In the first aspect disclosed in the present application, phenotypic and cytological analysis was performed to find id1The number of leaves in the mutant increased to twice that of the wild type. The apical meristem (SAM) of the wild type began to elongate and develop into the inflorescence meristem (IM) from the V4 stage, and continued to differentiate into the spikelet pair meristem (SPM), spikelet meristem (SM) and floret meristem (FM) as the development progressed. id1 The apical meristem of maize could not differentiate normally, and only leaf-like organs developed; the flowering period of maize was obtained by genetic analysis and positional cloning. Zm00001d032922 The gene, whose CDS sequence is shown in SEQ ID NO.1.

[0007] The second aspect disclosed in the present application relates to a flowering period regulating gene Zm00001d032922 The encoded protein has an amino acid sequence as shown in SEQ ID NO.2.

[0008] The third aspect disclosed in the present application relates to a method for amplifying the Zm00001d032922 The primer pair for the gene has sequences shown in SEQ ID NO.3 and SEQ ID NO.4.

[0009] The fourth aspect disclosed in the present application relates to an InDel marker for a phenotypic mutation in the flowering period of corn, based on the fourth version of the corn B73 reference genome, with an insertion of 27 bp (as shown in SEQ ID NO.5) and a deletion of 104 bp (as shown in SEQ ID NO.6) at the chr1.s_243204599 position, and an insertion of 12 bp (as shown in SEQ ID NO.7) at the chr1.s_243204603 position.

[0010] The fifth aspect disclosed in the present application relates to an InDel molecular marker for identifying phenotypic mutants, including a primer pair as shown in SEQ ID NO.8 and SEQ ID NO.9.

[0011] The sixth aspect disclosed in the present application relates to an identification Zm00001d032922 The overexpression material molecular marker includes the primer pair shown as SEQ ID NO.10 and SEQ ID NO.11.

[0012] In another aspect disclosed in the present application, the corn flowering stage regulating gene Zm00001d032922 , the amplification primers, the corn flowering period phenotypic mutation InDel site or the InDel molecular marker are used in the regulation of corn flowering period traits.

[0013] Another aspect disclosed in the present application relates to a SNP site that is significantly associated with the silking stage and pollen shedding stage of corn. Based on the fourth version of the corn B73 reference genome, C / T and A / T mutations exist at the chr1.s_243200783 and chr1.s_243205199 positions, respectively.

[0014] In another aspect disclosed in the present application, the corn flowering stage regulating gene Zm00001d032922 , the amplification primers, and the SNP sites are used in the breeding of inbred lines for corn silking and pollination stage traits.

[0015] Another aspect disclosed in the present application is a method for breeding a corn variety / line with an excellent haploid type of the Zm00001d032922 gene, comprising the following steps: (1) Determine by separate testing Zm00001d032922 The genotypes of the chr1.s_243205199 in the promoter region and the chr1.s_243200783 in the 3′UTR region of the gene; (2) In the maize inbred line population, the genotype with chr1.s_243205199 as A and chr1.s_243200783 as T was selected generation by generation to obtain the optimal haplotype.

[0016] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: 1. Positional Cloning Zm00001d032922 Genetic regulation of corn into reproductive growth affects the early or late flowering, and has important application value for breeding and improvement of corn during the silking and pollen shedding stages.

[0017] 2. Zm00001d032922 The identification of excellent haplotypes and analysis of their utilization pathways provide technical support and excellent germplasm resources for the utilization of this gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 In one embodiment of this application id1 Phenotypic analysis of mutants; in the figure, A: plant phenotype at the adult stage; B: statistical comparison of leaf numbers between the wild type and mutants; C: comparison of tassels at different developmental stages between the wild type and mutants; D: comparison of paraffin sections of tassels at different developmental stages between the wild type and mutants.

[0019] Figure 2 In one embodiment of this application Zm00001d032922 ( ID1 ) gene map cloning and gene structure; in the figure, A: Zm00001d032922 Positional cloning of genes; B: Zm00001d032922 Schematic diagram of the gene structure; C: Zm00001d032922Mutation site identification marker; D: Zm00001d032922 Schematic diagram of protein structure; E: Zm00001d032922 Function backfill verification.

[0020] Figure 3 This is a natural population verification diagram of the mutation site in an embodiment of the present application; the red frame in the figure shows the bands of the wild type and mutant marked by the InDel molecule, and the right side of the frame shows 88 natural populations in Zm00001d032922 ( ID1 ) Verification of the chr1.s_ 243204599 and chr1.s_ 243204603 sites on the gene.

[0021] Figure 4 This is the result of the correlation analysis between the silk-spinning period and the powder-shedding period in one embodiment of the present application; in the figure, the upper Zm00001d032922 Manhattan plot of the association analysis between SNPs and silking and pollination period traits; in the middle is Zm00001d032922 Schematic diagram of the gene structure; below is the LD block diagram of the SNP; the left picture is the pollination stage, and the right picture is the silking stage. DETAILED DESCRIPTION

[0022] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; the reagents and materials involved are all conventional commercial products unless otherwise specified; the tests and detection methods involved (such as genomic DNA extraction, RNA extraction and reverse transcription, agarose gel electrophoresis, corn paraffin sections, etc.) are all conventional methods unless otherwise specified.

[0023] Embodiment 1: id1 Phenotypic analysis of mutants id1 The mutant (Tang Jihua, collected in the Yuanyang Science and Education Park of Henan Agricultural University in 2018) is a reproductive conversion mutant discovered during field breeding selection. Observation of wild-type and mutant plants in the segregated population revealed that compared with the wild-type, id1 The mutant was unable to undergo reproductive transformation to differentiate male and female ears, and its leaf number was almost twice that of the wild type (see Figure 1 AB). Observation of male spikes at different developmental stages using stereomicroscopes and paraffin sections revealed that the apical meristem of the mutant was stagnant (see Figure 1 CD).

[0024] Embodiment 2: Zm00001d032922 Map-based cloning and functional verification of genes ID1 / id1 The offspring segregation population of heterozygous single plants was used as material. Through genetic analysis and map-based cloning, the target gene was located in a physical interval of about 315 kb on chromosome 1 of maize (see Figure 2 A).

[0025] The linkage markers and their sequences used in the map-based cloning process are shown in Table 1. Gene annotation and sequencing analysis within the candidate segment showed that Zm00001d032922 The gene (CDS sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) is a potential candidate gene, which contains 4 exons. id1 middle Zm00001d032922 The gene has a 12bp / 27bp insertion and a 104bp deletion at the +35bp position of the first exon, which causes the amino acid it encodes to shift frame and terminate prematurely (see Figure 2 BD).

[0026] Zm00001d032922 The gene cloning method is as follows: 1. Use the Plant Polysaccharide and Polyphenol RNA Extraction Kit to extract RNA from young leaves of mutants and wild-type plants at V4 stage according to the kit instructions, fully dissolve the total RNA with RNase-free double distilled water, and use DNaseI to remove possible DNA residues.

[0027] 2. Use agarose gel electrophoresis to preliminarily test the RNA quality. NanoDrop instrument detects RNA concentration and RNA absorbance at 260 nm and 280 nm. 260 / 280 Whether it is between 1.8 and 2.2 (28S: 18S>1.0) can determine the integrity and purity of the RNA.

[0028] 3. Use the obtained RNA as a template for reverse transcription, and the obtained cDNA will be packaged and stored in a -20℃ refrigerator for later use.

[0029] 4. Based on the maize B73 reference genome Zm00001d032922 Transcript sequence, design amplification primers: F: 5'- ATGCAGATGATGATGCTCTC- 3' (SEQ ID No. 3); R: 5'-CTAGAAGTTGTGGCTCCAG-3' (SEQ ID No. 4).

[0030] To verify this candidate gene and further clarify its biological function, a Zm00001d032922 The overexpression vector was used to transform maize embryos and obtain transgenic seeds. id1 The hybridization was performed and the F 1 The genotype of the ears was identified and the overexpression positive and id1 The seeds heterozygous for the mutation site were used for self-pollination to obtain F 2 , for F2 Separate the population for phenotypic observation and genotypic identification. id1 The mutation site is - / - When the transgenic positive plants showed normal reproductive transformation, the transgenic negative plants showed mutant phenotypes. Zm00001d032922 Can be covered id1 The germline mutant phenotype is produced id1 Functional genes for germline mutant phenotypes (see Figure 2 E) and named Zm00001d032922 .

[0031] Table 1 Zm00001d032922 Primers and sequences used for cloning .

[0032] Example 3: Corn Zm00001d032922 Development of molecular markers for mutation sites against Zm00001d032922 Develop easy-to-detect PCR molecular markers InDel for insertion and deletion, as follows: id1 The actual position of the mutation site in the fourth version of the maize B73 reference genome is 243204599 and 243204603 of chromosome 1; the following InDel primers were designed: InDel-F: 5' -TAGCTAGCGATCGACGACAGA -3' (SEQ ID No. 8); InDel-R: 5' -GGCTGGCTTCTCTTCCTCTT- 3' (SEQ ID No. 9).

[0033] against id1 Molecular marker detection of mutation sites includes the following steps: ① The reaction system is: DNA template (30-50 ng / μL) 1 μL; Left primer (10 μM) 0.5 μL; Right primer (10μM) 0.5μL; dH 2 O 2 μL; 2×PCR Mix 5μL.

[0034] ② The PCR amplification program was as follows: 1 cycle of pre-denaturation at 95℃ for 3 min; 35 cycles of denaturation at 95℃ for 15 s, annealing at 60℃ for 20 s, and extension at 72℃ for 20 s; Delay at 72℃ for 5 min; Soak at 25℃.

[0035] When examined on a 2% agarose gel, the mutant material appeared as a smaller band (see Figure 3 ).

[0036] Embodiment 4: Zm00001d032922 Candidate gene association analysis To clarify Zm00001d032922 The utilization pathway of the gene was to use 103 SNPs (single nucleotide polymorphism sites) (B73V4: Chr1: 243201405-243204865) of the gene in 471 inbred lines as genotypes, and candidate gene association analysis was performed based on the reproductive period phenotype, population structure (Q) and kinship (K) of the population. p <9.71E-03 (1 / 103) was used as the threshold, and a total of 22 SNPs were detected that were significantly associated with heading time (HD), pollen shed (PS), silking time (ST), and leaf number above ear (LANE) (see Table 2, Figure 4 ).

[0037] Table 2 Zm00001d032922 Results of candidate gene association analysis .

[0038] Embodiment five: Zm00001d032922 Excellent haplotype identification Among the 22 significantly associated SNPs mentioned above, except for intronic and synonymous mutations, Zm00001d032922 The chr1.s_243200783 (C / T) in the 3'UTR of the gene and the chr1.s_243205199 (A / T) in the promoter region were most significantly associated with the silking stage and pollen shedding stage traits ( p <10 -4 ,See Figure 4). Haplotype analysis based on these two SNPs revealed four combinations (Hap1 AC, Hap2 TC, Hap3 AT, Hap4 TT) in 483 inbred lines (see Table 2): Hap1 (244 copies), Hap2 (269 copies), Hap3 (26 copies), and Hap4 (8 copies). Among them, Hap3 was the optimal haplotype for the traits of tasseling period, pollen shedding period, silking period and number of leaves on ear, with an early growth period and a low distribution frequency in inbred lines (26 / 483). It was a rare haplotype and had utilization potential in breeding and improvement. Hap1 was an unfavorable haplotype for the traits of tasseling period, pollen shedding period, silking period and number of leaves on ear, with a high distribution frequency in inbred lines (244 / 484). The traits of tasseling period, pollen shedding period, silking period and number of leaves on ear of the vast majority of inbred lines with Hap2 and the very few inbred lines with Hap4 were between Hap1 and Hap3 (Table 3).

[0039] Table 3 Haplotype analysis .

[0040] Although some preferred embodiments of the present application have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0041] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations to the present invention fall within the scope of the claims of the present application and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A gene regulating flowering time in maize Zm00001d032922 , and its nucleotide sequence is shown in SEQ ID NO.

1.

2. A gene regulating flowering time in maize Zm00001d032922 The encoded protein has an amino acid sequence as shown in SEQ ID NO.

2.

3. A maize flowering period phenotypic mutation InDel marker, based on the fourth version of the maize B73 reference genome, has an insertion of 27 bp as shown in SEQ ID NO.5 and a deletion of 104 bp as shown in SEQ ID NO.6 at the chr1.s_243204599 position, and an insertion of 12 bp as shown in SEQ ID NO.7 at the chr1.s_243204603 position.

4. Identification id1 The InDel molecular marker of the mutant includes the primer pair shown as SEQ ID NO.8 and SEQ ID NO.

9.

5. The maize flowering stage regulating gene according to claim 1 Zm00001d032922 , the use of the corn flowering period phenotypic mutation InDel marker as described in claim 3, and the InDel molecular marker as described in claim 4 in the regulation of flowering period and / or corn male and female ear development traits.

6. A SNP marker significantly associated with corn flowering period, based on the fourth version of the corn B73 reference genome, with C / T and A / T mutations at chr1.s_243200783 and chr1.s_243205199, respectively.

7. The maize flowering stage regulating gene according to claim 1 Zm00001d032922 , the use of the corn flowering period phenotypic mutation InDel marker according to claim 3, the InDel molecular marker according to claim 4, and the SNP marker according to claim 6 in the breeding of corn flowering period trait inbred lines.

8. A method for breeding a corn variety / line with an excellent haploid gene of Zm00001d032922, comprising the following steps: (1) Determine by separate testing Zm00001d032922 The genotypes of the chr1.s_243205199 in the promoter region and the chr1.s_243200783 in the 3′UTR region of the gene; (2) In the maize inbred line population, the genotype with chr1.s_243205199 as A and chr1.s_243200783 as T was selected generation by generation to obtain the optimal haplotype.

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