DNA SEQUENCE FOR REGULATING MAIZE LEAF ANGLE, AND MUTANT, MOLECULAR MARKERS, DETECTION PRIMERS, AND USE THEREOF

Abstract

A key DNA sequence for regulating a maize leaf angle and a mutant thereof are provided, which have polynucleotide sequences shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. The DNA sequence for regulating a maize leaf angle and the mutant thereof provided by present disclosure can regulate the expression of ZmNAC16 gene in a maize pulvinus, and thus can be used for the improvement of maize leaf angle and plant type and further for the cultivation of new maize varieties. The present disclosure further provides specific detection primers for detecting mutations of the DNA key sequence and the mutant, and detection primers for detecting an expression level of ZmNAC16 gene in maize. These detection primers can be used to directionally improve a maize leaf angle and also shows application potential for breeding of dense-planting-tolerant and high-yield maize.

Claims

1. A key DNA sequence for regulating a maize leaf angle, wherein the key DNA sequence has a polynucleotide selected from the group consisting of (a), (b), (c), and (d): (a) a first polynucleotide sequence shown in SEQ ID No. 1; (b) a second polynucleotide, wherein the second polynucleotide hybridizes with a complementary sequence of SEQ ID No. 1 under stringent hybridization conditions, wherein a protein encoded by the second polynucleotide still has a function of regulating the maize leaf angle; (c) a third polynucleotide having at least 90% or more homology with the first polynucleotide sequence shown in SEQ ID No. 1; and (d) a polynucleotide mutant obtained through deletion, substitution, or insertion of one or more bases on the basis of the first polynucleotide sequence shown in SEQ ID No. 1, wherein a protein encoded by the polynucleotide mutant still has the function or an activity of regulating the maize leaf angle.

2. A mutant of the key DNA sequence for regulating the maize leaf angle according to claim 1, wherein the mutant has a polynucleotide sequence shown in SEQ ID No. 2.

3. A recombinant expression vector, wherein the recombinant expression vector carries the key DNA sequence according to claim 1, or the recombinant expression vector carries a mutant of the key DNA sequence, and the mutant has a polynucleotide sequence shown in SEQ ID No. 2.

4. A molecular marker for regulating an expression of ZmNAC16 gene in a maize pulvinus, wherein the molecular marker comprises SNP_3_6945310_C/T, Indel_3_6945248_C/CT, and Indel_3_6945836_T/TTGCA.

5. A specific detection primer for detecting mutations of the molecular marker according to claim 4, wherein the specific detection primer has nucleotide sequences shown in SEQ ID No. 3 and SEQ ID No. 4.

6. Use of the key DNA sequence according to claim 1 in a regulation of the maize leaf angle, or in a cultivation of a high-yield maize variety or a dense-planting-tolerant maize variety, wherein the regulation of the maize leaf angle comprises reducing the maize leaf angle.

7. Use of the mutant according to claim 2 in a regulation of the maize leaf angle, or in a cultivation of a high-yield maize variety or a dense-planting-tolerant maize variety, wherein the regulation of the maize leaf angle comprises reducing the maize leaf angle.

8. Use of the molecular marker according to claim 4 in a regulation of the expression of ZmNAC16 gene in maize.

9. A specific primer pair for detecting ZmNAC16 gene, wherein the specific primer pair has polynucleotides shown in SEQ ID No. 5 and SEQ ID No. 6.

10. A method for cultivating a high-yield maize variety or a dense-planting-tolerant maize variety, comprising: using the key DNA sequence according to claim 1 or a mutant of the key DNA sequence to increase an expression level of ZmNAC16 gene in a maize pulvinus, wherein the mutant has a polynucleotide sequence shown in SEQ ID No. 2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1A shows the significant correlation of the three variation sites in the ZmNAC16 intron and 3′-UTR region with the flowering period and leaf number phenotype of maize obtained by the genome-wide association study (GWAS) method, where the three mutation sites are SNP_3_6945310_C/T, Indel_3_6945248_C/CT, and Indel_3_6945836_T/TTGCA and the red arrow parallel to the X-axis in the figure represents ZmNAC16; and FIG. 1B shows the gene structure of ZmNAC16 and the location information of the three mutation sites SNP_3_6945310_C/T, Indel_3_6945248_C/CT, and Indel_3_6945836_T/TTGCA.

[0051] FIG. 2 shows the mutations of 16 different groups of inbred lines at the three mutation sites (SNP_3_6945310_C/T, Indel_3_6945248_C/CT, and Indel_3_6945836_T/TTGCA), where overall, the three sites SNP_3_6945310_C/T, Indel_3_6945248_C/CT, and Indel_3_6945836_T/TTGCA are linked together to form two haplotypes: Hap1_0/C/0 and Hap2_1/T/4.

[0052] FIG. 3A and FIG. 3B show the leaf angle phenotype comparison of inbred lines of different mutation types at the three variant sites in the ZmNAC16 intron and 3′-UTR region, where the Hap1_0/C/0 and Hap2_1/T/4 represent the two genotypes in FIG. 2, respectively; and FIG. 3C shows the expression analysis of the ZmNAC16 gene in pulvini of folded and unfolded leaves of the two inbred lines Hap1_0/C/0 and Hap2_1/T/4 at the V7 stage, where the ZmNAC16 gene is remarkably highly expressed in pulvini of unfolded leaves.

[0053] FIG. 4A shows the statistical analysis of corresponding phenotypes of the two allelic mutations of Hap1_0/C/0 and Hap2_1/T/4; and FIG. 4B shows the selection analysis of Hap1_0/C/0 and Hap2_1/T/4 in a maize breeding process, where colors corresponding to different allelic mutations are the same as in FIG. 4A and Hap1_0/C/0 is obviously artificially selected in the maize breeding process.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] The present disclosure will be further described below in conjunction with specific examples, and the advantages and features of the present disclosure will become clearer from the description. However, these examples are only exemplary and do not constitute any limitation to the scope of the present disclosure. Those skilled in the art should appreciate that modifications and substitutions can be made to the details and forms of the present disclosure without departing from the spirit and scope of the present disclosure, but these modifications and substitutions fall within the protection scope of the present disclosure.

[0055] For the inbred lines used in the following examples, relevant information can be obtained and corresponding seeds can be applied from the “Chinese Crop Germplasm Resources Information System”.

Example 1 Mutation Sites in the ZmNAC16 Intron and 3′-UTR Region Regulated a Maize Leaf Angle by Controlling an Expression Level of the ZmNAC16 Gene

[0056] 1. Discovery of the Mutation Sites in the ZmNAC16 Intron and 3′-UTR Region

[0057] With deep (>10×) resequencing data of 350 maize inbred lines in combination with the published maize B73 V3 genome, 25,320,664 single-nucleotide polymorphism molecular markers (SNPs) and 4,319,510 indel polymorphism molecular markers (Indel) were discovered. These discovered molecular markers were used to estimate the population structure and genetic relationship of the 350 maize inbred lines, and then in combination with collected leaf angle phenotypes of 4 environments, GWAS was conducted. One SNP marker SNP_3_6945310_C/T and two Indel markers Indel_3_6945248_C/CT and Indel_3_6945836_T/TTGCA on chromosome 3 were found to be significantly associated with the maize leaf angle trait (FIG. 1). Further research showed that the three mutation sites were located in the intron and 3′-UTR (SEQ ID No. 2) region of ZmNAC16.

[0058] 2. Acquisition of a Nucleotide Sequence of a Genomic Region where the Mutation Sites in the ZmNAC16 Intron and 3′-UTR Region were Located

[0059] Specific amplification primers were designed based on the B73 V3 genome sequence of the region where SNP_3_6945310_C/T, Indel_3_6945248_C/CT, and Indel_3_6945836_T/TTGCA were located, and amplification was conducted for 6 different types of maize inbred lines to obtain the nucleotide sequence of this region and the accurate information about the mutation (FIG. 2).

[0060] 3. Regulation of the ZmNAC16 Gene Expression by the Mutation Sites in the ZmNAC16 Intron and 3′-UTR Region

[0061] According to the genotypes at the three mutation sites SNP_3_6945310_C/T, Indel_3_6945248_C/CT, and Indel_3_6945836_T/TTGCA, the sequenced inbred lines were divided into Hap1_0/C/0 and Hap2_1/T/4 (FIG. 2 and FIG. 3). The two types of representative inbred lines (FIG. 2) were planted in the field, separately. When there were 7 fully unfolded leaves (V7 stage), pulvini (a part where a leaf and a leaf sheath are connected) of fully unfolded leaves (V7 leaves) and folded leaves (V5 leaves) were sampled and quick-frozen with liquid nitrogen. Pulvini of every 5 individual plants were mixed into a sample, and 3 biological replicates were set for each inbred line. Then RNA was extracted by the TRIzol method, and the expression level of the ZmNAC16 gene was detected using specific primers (SEQ ID No. 5 and SEQ ID No. 6). It was found that the ZmNAC16 gene was significantly highly expressed in the leaves of the Hap2_1/T/4 inbred line (FIG. 3), indicating that the mutation sites in the ZmNAC16 intron and 3′-UTR region could regulate the expression of the ZmNAC16 gene in maize leaves.

Example 2 the Mutation Sites in the ZmNAC16 Intron and 3′-UTR Region were Subjected to Strong Artificial Selection in the Modern Maize Breeding Process

[0062] In the early stage of this experiment, 350 maize inbred line materials from different breeding periods in China and the United States were collected, including 163 breeding materials from the early period (Public-US) and the modern period (Ex-PVP) of the United States, and 187 main maize breeding materials from the early period (CN1960&70s), middle period (CN1980&90s), and current period (CN2000&10s) of China. The frequency distribution of the two genotypes Hap1_0/C/0 and Hap2_1/T/4 of ZmNAC16 in these materials was analyzed, and it was found that, with the advancement of the breeding period, the frequency of Hap1_0/C/0 had increased significantly in the maize breeding processes of China and the United States, indicating that the Hap1_0/C/0 genotype was subjected to strong artificial selection in the modern maize breeding process. Phenotypic analysis showed that Hap1_0/C/0 could significantly reduce the maize leaf angle, and it was consistent with the fact that the compact plant type is a key target for breeding of dense-planting-tolerant maize, which further confirmed that the mutation sites in the ZmNAC16 intron and 3′-UTR region were subjected to strong artificial selection in the modern maize breeding process.