Corn grain length and hundred-grain weight associated SNP marker, dCAPS primer and application of dCAPS primer

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker associated with corn grain length and hundred-grain weight, a dCAPS primer and application of the dCAPS primer. According to the functional marker developed on the basis of SNP sites (chr9.s121100593 and chr9.s121100040) existing in the ZmGWT1a gene, haplotype analysis and identification and creation of a corn high-yield breeding material are carried out, the breeding period of new crop varieties is shortened, the breeding cost is reduced, the breeding efficiency is improved, and a technical approach is provided for development and utilization of the ZmGWT1a gene.

Description

[0001] This invention is a divisional application of the patent application with the application number 2023101451271, the application date of February 21, 2023, and the invention title of "Maize Kernel Development Regulation Gene ZmGWT1a , Its Encoded Protein, SNP Locus, Functional Marker and Their Applications". Technical Field

[0002] This invention relates to the field of maize assisted molecular breeding technology, and specifically relates to an SNP marker, dCAPS primer related to maize kernel length and 100-kernel weight, and their applications. Background Art

[0004] Maize kernel size is an important factor in maize yield. Among them, kernels, as the decisive factor in maize yield, have become the focus of attention. As the main organ for maize yield, the molecular mechanism of its formation and development has received extensive attention. At present, although many genes regulating maize kernel development and filling have been cloned, due to the extremely complex biological process of maize embryo and endosperm development, the key genes and molecular mechanisms for precisely regulating kernel filling still need to be further analyzed.

[0005] On the other hand, glycosylphosphatidylinositol (GPI) modification is an important post-translational modification in eukaryotes. GPI-anchored proteins (GPI-APs) are involved in various biological processes such as transmembrane signal transduction, cell surface protection, cell adhesion, and cell wall formation. Research has shown that mutations in genes related to GPI biosynthesis, anchoring, and remodeling can lead to various physiological and reproductive defects in mammals. However, the mechanism of action of genes related to GPI synthesis and regulation in plants is still unclear.

[0006] The information disclosed in this background art section is only used to deepen the understanding of the background art of this disclosure, and should not be regarded as an admission or any form of implication that this information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0007] This invention uses a natural mutant of maize kernels discovered during the process of selecting inbred lines in maize field breeding gwt1a to clone the key gene affecting GPI synthesis ZmGWT1a , which encodes an acyltransferase in the 4th step of the glycosylphosphatidylinositol (GPI) synthesis pathway. This gene ZmGWT1a regulates maize embryo and endosperm development, affects the accumulation of storage substances in kernels, but does not affect the normal growth and development of plants, and has important value for increasing maize yield.

[0008] In the first aspect of this disclosure, through phenotypic and cytological analysis, it is found that gwt1aThe 100-kernel weight of mutant kernels was reduced by 49.7% compared to the wild type, and the embryo and endosperm development were abnormal, with reduced proliferation in the cells of the endosperm basal transfer layer; the ZmGWT1a gene affecting maize kernel development was isolated by genetic analysis and map-based cloning, and its CDS sequence is shown in SEQ ID NO.1.

[0009] In a second aspect of the present disclosure, there is provided an encoded protein of a gene ZmGWT1a regulating grain development, and its amino acid sequence is shown in SEQ ID NO.2.

[0010] In a third aspect of the present disclosure, there is provided a primer pair for amplifying the ZmGWT1a gene, and its sequences are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0011] In a fourth aspect of the present disclosure, there is provided a SNP marker for maize kernel phenotypic mutation, based on the fourth version of the maize B73 reference genome, with a G / A mutation at the position chr9.s_121097330.

[0012] In a fifth aspect of the present disclosure, there is provided a dCAPS primer for identifying phenotypic mutants, including primer pairs shown in SEQ ID NO.5 and SEQ ID NO.6; and / or primer pairs shown in SEQ ID NO.7 and SEQ ID NO.8.

[0013] In yet another aspect of the present disclosure, the maize grain development regulatory gene ZmGWT1a , the amplification primer, the maize kernel phenotypic mutation SNP marker or the dCAPS primer are applied to the regulation of grain size and / or maize embryo and endosperm development traits.

[0014] In another aspect of the present disclosure, there is provided a SNP marker associated with maize grain length and 100-kernel weight, based on the fourth version of the maize B73 reference genome, with a C / T mutation at the position chr9.s_121100040 or / and chr9.s_121100593; and it can be applied to ZmGWT1a excellent haplotypes of genes.

[0015] In still another aspect of the present disclosure, there is provided a dCAPS primer for identifying excellent haplotypes of genes ZmGWT1a , including primer pairs shown in SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, and / or SEQ ID NO.13 and SEQ ID NO.14.

[0016] In yet another aspect of the present disclosure, the maize grain development regulatory gene ZmGWT1a, The amplification primer, the SNP marker, and the dCAPS primer are applied in the breeding of maize grain size varieties / lines.

[0017] One or more technical solutions provided in the embodiments of the present disclosure have at least any one of the following technical effects or advantages: 1. The gene cloned by map-based cloning ZmGWT1a regulates the development of maize embryos and endosperms, affects the accumulation of storage substances in grains, but does not affect the normal growth and development of plants, and is of great value for increasing maize yield.

[0018] 2. ZmGWT1a Identification of excellent haplotypes and analysis of utilization pathways provide technical support and excellent germplasm resources for the utilization of this gene. Brief Description of the Drawings

[0019] Figure 1 In one embodiment of the present disclosure ZmGWT1a mutant ( gwt1a ) grain phenotype analysis; in the figure, A: grain phenotype on the segregating ear; B: comparison of wild-type and mutant grains on the same ear; C: comparison of 100-grain weight of wild-type (WT) and mutant gwt1a ; D: comparison of longitudinal sections of wild-type and mutant grains; E: difference in germination of wild-type and mutant grains; F: difference in germination rate of wild-type and mutant grains; G: comparison of above-ground parts of wild-type and mutant at 10 days after sowing; H: comparison of roots of wild-type and mutant at 10 days after sowing; I: comparison of plants of wild-type and mutant at the adult stage; J: comparison of self-pollinated ears of homozygous wild-type (+ / +) and mutant (- / -) plants.

[0020] Figure 2 Cytological analysis of wild-type and mutant grains in one embodiment of the present disclosure; in the figure, A: comparison of paraffin sections of mutant and wild-type grains at 10 - 18 days after pollination; B: comparison of embryos of mutant and wild-type grains at 18 days after pollination; C: transmission electron microscopy observation of endosperms of mutant and wild-type; D: comparison of basal transfer layers of mutant and wild-type endosperms.

[0021] Figure 3 In one embodiment of the present disclosure ZmGWT1a ( gwt1a ) map-based cloning, gene structure and function verification of the gene; in the figure, A: ZmGWT1a map-based cloning of the gene; B: ZmGWT1a structural schematic diagram of the gene; C: structural schematic diagram of the ZmGWT1a protein; D - E: gwt1a allele verification with ZmGWT1a gene premature termination mutant.

[0022] Figure 4Molecular marker diagrams of dCAPS1-F1 / R1 and dCAPS1-F1 / R1 and their digested products in an embodiment of the present disclosure; in the figure, A: gwt1a Phenotypes of mature wild-type (WT) grains and mutant grains; B: Agarose gel electrophoresis bands of dCAPS1-F1R1 PCR products; C: Agarose gel electrophoresis bands of dCAPS1-F2R2 PCR products; D: After the dCAPS1-F2R2PCR products are Sma digested with I enzyme, the electrophoresis bands in a 10% nucleic acid PAGE gel.

[0023] Figure 5 Results of grain length association analysis in an embodiment of the present disclosure; in the figure, the upper part is ZmGWT1a Manhattan plot of SNP and grain length trait association analysis; the middle part is ZmGWT1a Schematic diagram of the gene structure; the lower part is the LD block diagram of SNP.

[0024] Figure 6 Molecular markers for haplotype utilization in an embodiment of the present disclosure; in the figure, A: Agarose gel electrophoresis bands of dCAPS2-F1R1 PCR products; B: Agarose gel electrophoresis bands of dCAPS2-F2R2 (chr9.s_121100040) PCR products; C: After the dCAPS2-F2R2 PCR products are Hind digested with III enzyme, the electrophoresis bands in a 4% nucleic acid agarose gel; D: Agarose gel electrophoresis bands of dCAPS2-F3R3 (chr9.s_1211005593) PCR products; E: After the dCAPS2-F2R2 PCR products are Nde digested with I enzyme, the electrophoresis bands in a 4% nucleic acid agarose gel; Note: Lanes 1 (LY042) and 2 (CIMBL98) are Hap3; lanes 3 (CF3) and 4 (R08) are Hap2; lanes 5 (BS16) and 6 (MN) are Hap1. Detailed implementation manners

[0025] In the following embodiments, the instruments and equipment involved are all conventional instruments and equipment unless otherwise specified; the reagents and materials involved are all commercially available conventional products unless otherwise specified; the test and detection methods involved (such as extraction of genomic DNA, extraction and reverse transcription of RNA, RT-PCR, real-time fluorescence quantitative PCR, BN-PAGE gel electrophoresis, Westen blot, paraffin section of maize grains, etc.) are all conventional methods unless otherwise specified.

[0026] Example 1: gwt1a Phenotypic analysis of mutants gwt1aMutants (collected by Tang Jihua, 2018, Yuanyang Science and Education Park, Henan Agricultural University) are grain mutants discovered during field breeding and selection. Observation of mature wild-type and mutant grains on the same ear revealed that, compared with the wild-type, gwt1a The mutant grains became smaller, the endosperm was not fully filled, and the top was slightly collapsed, resulting in a significant decrease in the 100-grain weight of the mutant (50.3% of the wild type) (see Figure 1 AD). The mature mutant grains can germinate normally, and the mutants can grow into plants and self-fertilize after germination (see Figure 1 GJ, but its bud rate (91.6%) was lower than that of the wild type (99.6%) (see Figure 1 EF).

[0027] 10~18 days after pollination gwt1a Paraffin sections of mutant and wild-type kernels were observed and it was found that the mutant embryo developed normally, the proliferation of cells in the endosperm basal transfer layer was reduced, and the filling of endosperm starch granules and protein bodies was reduced compared with the wild-type (see Figure 2 ).

[0028] Embodiment 2: ZmGWT1a Map-based cloning and functional verification of genes use gwt1a The F2 segregating population of the maize gene was used as material by genetic analysis and positional cloning. The target gene was located in the physical interval of about 789 kb on chromosome 9 of maize (see Figure 3 A).

[0029] 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 Zm00001d047181 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, containing 16 exons. gwt1a )middle Zm00001d016902 The gene has a single base mutation from G to A at +933bp in exon 12, causing the 311th amino acid encoded by it to mutate from tryptophan to a stop codon (see Figure 3 B).

[0030] Zm00001d047181 The gene cloning method is as follows: 1) Use the Plant Polysaccharide and Polyphenol RNA Extraction Kit to extract RNA from mutant and wild-type endosperm 10 days after pollination according to the kit instructions, fully dissolve the total RNA with RNase-free double distilled water, and use DNaseI to remove possible DNA residues.

[0031] 2) RNA quality was initially tested by agarose gel electrophoresis. The RNA concentration and the absorbance of RNA at 260 nm and 280 nm were tested by NanoDrop instrument. 260 / 280 Whether it is between 1.8 and 2.2 (28S: 18S>1.0) can determine the integrity and purity of the RNA.

[0032] 3) The obtained RNA was used as a template for reverse transcription, and the obtained cDNA was packaged and stored in a -20°C refrigerator for later use.

[0033] 4) Based on the maize B73 reference genome Zm00001d047181 Transcript sequence, design amplification primers: F: 5'-ATGGAGGGGCCGCCTCTT-3' (SEQ ID No. 3); R: 5'-TCACCAGAATTTCATCCTAACACC-3' (SEQ ID No. 4).

[0034] To verify the candidate gene, we purchased the EMS mutant from the maize mutant library (http: / / elabcaas.cn / memd / ). Zm00001d047181 Premature termination mutant material ( gwt1a-2 ). Change + / gwt1a With + / gwt1a-2 The hybridization was carried out, and the phenotype and genotype of the F1 ears were observed. The results showed that the wild-type and mutant grains on the F1 ears met the Mendelian segregation ratio of 3:1, and only the mutant phenotype grains contained two mutation sites at the same time. Zm00001d047181 is produced gwt1a The functional gene of the kernel mutant phenotype is named ZmGWT1a .

[0035] Table 1 ZmGWT1a Primers and sequences used for cloning .

[0036] Example 3: Corn ZmGWT1a Development of molecular markers for mutation sites against ZmGWT1a The single base mutation of dCAPS1 was developed to be easily detected by PCR molecular markers, as follows: gwt1a The actual position of the mutation site in the fourth version of the maize B73 reference genome is 121,097,330, and the following dCAPS primers were designed: dCAPS1-F1: 5'-GTTCTCCTGCACGTTCGATA-3' (SEQ ID No. 5); dCAPS1-R1: 5’-CGTACCATTCGACGAGAAACT-3’ (SEQ ID No.6); dCAPS1-F2: 5’-CTCCCATGGCAGGATCCCG-3’ (SEQ ID No.7); dCAPS1-R2: 5’ –TGACTTGAGTGTTCCGGTTCTTGC-3’ (SEQ ID No.8).

[0037] Among them, dCAPS1-F1R1 is the outer primer pair, which can specifically amplify ZmGWT1a gene sequence (see Figure 4 B); dCAPS1-F2R2 is the inner primer pair, and the second-round amplification is carried out using the PCR product diluted 10 times of dCAPS1-F1R1 as the template.

[0038] For gwt1a the molecular marker detection of mutants, it includes the following steps: (1) The reaction system is: 1 μL of DNA template (30 - 50 ng / μL); 0.5 μL of Left primer (10 μM); 0.5 μL of Right primer (10 μM); 2 μL of ddH2O; 5 μL of 2×PCR Mix.

[0039] (2) The PCR amplification program of dCAPS1-F1R1 is: 1 cycle of pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 20 s, and extension at 72°C for 30 s; Delay at 72°C for 5 min; Soak at 25°C.

[0040] (3) The PCR amplification program of dCAPS1-F2R2 is: 1 cycle of pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 20 s, and extension at 72°C for 15 s; Delay at 72°C for 5 min; Soak at 25°C.

[0041] The PCR product of dCAPS1-F2R2 is diluted 10 times, digested with restriction endonuclease Sma Ⅰ, and then detected with 10% nucleic acid PAGE gel. After digestion, the wild-type material shows a lower band (see Figure 4 D).

[0042] Example 4: ZmGWT1a Candidate gene association analysis To clarify ZmGWT1aUtilization pathways of the gene, using 227 SNPs (single nucleotide polymorphism sites) (B73V4: 121087724 - 121104200) in 507 inbred lines as genotypes, and performing candidate gene association analysis based on the grain phenotypes, population structure (Q), and genetic relationship (K) of this population. Located at ZmGWT1a chr9.s_121092753 on the second exon of the gene, chr9.s_121093053, chr9.s_121093298, chr9.s_121093370, chr9.s_121094277, chr9.s_121095184, chr9.s_121097032 in the intron, and chr9.s_121100040 and chr9.s_121100593 in the 3’UTR were significantly associated with grain length and 100 - grain weight traits (p < 4.41E - 03, see Figure 5 ).

[0043] Example 5: ZmGWT1a Identification of excellent haplotypes Among the significantly associated sites of grain length and 100 - grain weight, chr9.s_121092753, chr9.s_121093053, chr9.s_121094277, chr9.s_121095184, chr9.s_121097032 and chr9.s_121100593 were in complete LD. Therefore, subsequent haplotype analysis was only performed on chr9.s_121100593 and chr9.s_121100040 located in the 3’UTR. There were 3 combinations in 444 inbred lines, and the differences in grain length were extremely significant ( p = 9.18E - 07) (see Table 2): Hap1 (CC, 2 lines), Hap2 (CT, 410 lines), Hap3 (TC, 32 lines). Among them, the average grain length of the excellent haplotype Hap3 was 9.75 mm, and its distribution frequency in inbred lines was relatively low (32 / 444), belonging to a rare haplotype, which had potential for utilization in breeding improvement; the average grain length of the unfavorable haplotype Hap1 was 8.63 mm, and its distribution frequency in inbred lines was extremely low (2 / 444), belonging to a rare haplotype; the haplotype of most inbred lines was Hap2, with a high distribution frequency (410 / 444) and moderate grain length (see Table 2).

[0044] Table 2 Haplotype analysis .

[0045] Example 6: ZmGWT1a Development of molecular markers for excellent haplotypes Based on the identification of the haplotypes with excellent grain length, simple and easy-to-operate PCR markers dCAPS2 were developed for these two SNPs, and their actual positions in the fourth version of the maize B73 reference genome are 121,100,040 bp and 121,100,593 bp respectively.

[0046] dCAPS primers: dCAPS2-F1: 5’-GGTCGCTGCTGCATTAAAAGT-3’ (SEQ ID No.9); dCAPS2-R1: 5’ -TTGCCACAGAAAAGTCAAACAGA-3’ (SEQ ID No.10); dCAPS2-F2: 5’ -CTCAACTCTGAGCTGGAGCCCAAGCT-3’ (SEQ ID No.11); dCAPS2-R2: 5’ -GTGACGAGCTGGTGCCGTTT-3’ (SEQ ID No.12); dCAPS2-F3: 5’ -TGATAAATTTAATAATTTATTCATAT-3’ (SEQ ID No.13); dCAPS2-R3: 5’ -ATTCGGGTGCACGATCCG-3’ (SEQ ID No.14).

[0047] Among them, dCAPS2-F1R1 is the outer primer pair, which can specifically amplify the genomic sequence containing the two SNPs; dCAPS2-F2R2 and dCAPS2-F3R3 are the inner primer pairs for chr9.s_121100040 and chr9.s_1211005593 respectively, and the PCR product of dCAPS2-F1R1 diluted 10 times is used as the template for the second-round amplification.

[0048] The molecular marker detection for the excellent haplotypes includes the following steps: (1) The reaction system is: 1 μL of DNA template (30 - 50 ng / μL); 0.5 μL of Left primer (10 μM); 0.5 μL of Right primer (10 μM); 2 μL of ddH2O; 5 μL of 2×PCR Mix.

[0049] (2)The PCR amplification procedure of dCAPS1-F1R1 is as follows: 1 cycle of pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 20 s, and extension at 72°C for 30 s; Delay at 72°C for 5 min; Soak at 25°C.

[0050] (3)The PCR amplification procedure of dCAPS2-F2R2 is as follows: 1 cycle of pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 20 s, and extension at 72°C for 15 s; Delay at 72°C for 5 min; Soak at 25°C.

[0051] (4)The PCR amplification procedure of dCAPS2-F3R3 is as follows: 1 cycle of pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 52°C for 20 s, and extension at 72°C for 15 s; Delay at 72°C for 5 min; Soak at 25°C.

[0052] The PCR products of dCAPS2-F2R2 and dCAPS2-F3R3 are digested with restriction enzymes Hind III and Nde I respectively, and then detected by 4% nucleic acid agarose gel. Hap1 shows a higher band and a lower band respectively; Hap2 shows higher bands for both markers; Hap3 shows lower bands for both markers (see Figure 6 ).

[0053] Although some preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0054] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of its inventive concept. Thus, if these modifications and variations of the present disclosure fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. An SNP marker associated with the length of corn kernels and the weight of 100 kernels. Based on the fourth version of the maize B73 reference genome, there is a C / T mutation at the position of chr9.s_121100040 or / and chr9.s_121100593.

2. A method for identifying genes ZmGWT1a dCAPS primers for excellent haplotypes, including primer pairs shown in SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, and / or SEQ ID NO.13 and SEQ ID NO.

14.

3. The application of the SNP marker according to claim 1 or the dCAPS primer according to claim 2 in at least one of the following (1) to (7): (1) Identifying genes ZmGWT1a Haplotypes or reagents for preparing identifying genes ZmGWT1a Haplotypes; (2) Regulating the trait of corn kernel size or preparing a reagent for regulating the trait of corn kernel size; (3) Breeding varieties / lines with the trait of corn kernel size or preparing a reagent for breeding varieties / lines with the trait of corn kernel size; (4) Regulating the trait of corn kernel length or preparing a reagent for regulating the trait of corn kernel length; (5) Breeding varieties / lines with the trait of corn kernel length or preparing a reagent for breeding varieties / lines with the trait of corn kernel length; (6) Regulating the trait of 100-kernel weight of corn or preparing a reagent for regulating the trait of 100-kernel weight of corn; (7) Breeding varieties / lines with the trait of 100-kernel weight of corn or preparing a reagent for breeding varieties / lines with the trait of 100-kernel weight of corn.

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