Molecular marker for regulating the length of the main axis of the tassel of maize and its use

Abstract

This invention discloses a molecular marker for regulating the length of the maize tassel axis and its application, belonging to the field of biotechnology. The molecular marker is located on chromosome 7 of the maize reference genome (Zm-B73-REFERENCE-NAM-5.0), with a 19 bp deletion at 151,876,914-151,876,932 bp, a 40 bp insertion at 151,876,933 bp, and an 8 bp insertion at 151,877,050 bp, as shown in SEQ ID NO.1. Primers for amplifying the molecular marker are shown in SEQ ID NO.2 and SEQ ID NO.3. The molecular marker or the primers for amplifying it can be used to breed maize varieties with shorter tassel axes. This invention significantly improves breeding selection efficiency and has important application value and promising prospects for achieving ideal plant type goals.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a long molecular marker for regulating the main axis of maize tassels and its application. Background Technology

[0002] corn( Zea mays L. As a typical monoecious crop, the male tassel is not only an important component of the reproductive system but also one of the key agronomic traits determining yield potential. Previous studies have shown that male tassel size is negatively correlated with yield: the male tassel grows at the apex and has a certain apical dominance, so its development consumes a large amount of photosynthetic products and competes with the female ear for nutrients. At the same time, an overly developed male tassel may shade the upper functional leaves, thereby reducing the overall light energy utilization efficiency and thus affecting maize yield.

[0003] Appropriately shortening the length of the tassel's main axis not only helps reduce the plant's consumption of photosynthetic products but also improves light penetration and ventilation within the canopy, thereby further enhancing the canopy's light energy utilization efficiency and yield potential. Tassel main axis length, as a quantitative trait, exhibits multi-gene control and its phenotype is easily influenced by environmental factors, posing a challenge to the identification of relevant regulatory genes. With changing breeding objectives and continuous improvement of maize varieties, tassel morphology has undergone significant changes, with tassel length generally showing a gradual shortening trend. In particular, with the promotion of high-density planting technology and the demand for large-scale production, maize varieties with shorter tassel main axes have become one of the choices for high-density planting.

[0004] Therefore, using molecular biology techniques to analyze the genetic regulatory network of tassel main axis length and develop functional molecular markers closely linked to it can not only provide technical support for selecting maize varieties with shorter tassel main axes, but also accelerate the breeding process of high-yield and high-density-tolerant maize varieties. Summary of the Invention

[0005] Based on the above technical background, the main objective of this invention is to provide a molecular marker related to the length of the maize tassel main axis. The provided molecular marker can provide an effective molecular-assisted selection method for maize varieties with shorter tassel main axis lengths.

[0006] The technical solution of this invention is as follows: One of the objectives of this invention is to provide a candidate gene for regulating the genetic variation of maize tassel axis length. ZmTHX48 The gene is located on chromosome 7, 151,872,221-151,876,853 bp of the maize reference genome (Zm-B73-REFERENCE-NAM-5.0).

[0007] The second objective of this invention is to provide a molecular marker related to the length of the maize tassel main axis for selection of maize varieties with shorter tassel main axis lengths (28-32 cm).

[0008] Based on the above candidate genes ZmTHX48 The promoter design-related molecular marker is located on chromosome 7 of the maize reference genome (Zm-B73-REFERENCE-NAM-5.0), with a 19 bp deletion at 151,876,914-151,876,932 bp, a 40 bp insertion at 151,876,933 bp, and an 8 bp insertion at 151,877,050 bp. Its nucleotide sequence is shown in SEQ ID NO.1.

[0009] The third objective of this invention is to provide a primer for amplifying the aforementioned molecular marker.

[0010] Primers for amplifying the aforementioned molecular markers were developed, and the nucleotide sequences of the left primer F (as shown in SEQ ID NO.2) and the right primer R (as shown in SEQ ID NO.3) are as follows: Left primer F (SEQ ID NO.2): TACTGCTACTGCATCGGTCG; Right primer R (SEQ ID NO.3): GCGTTTCCGTTCCAACCCC.

[0011] The fourth objective of this invention is to provide an amplification system and procedure for PCR amplification of the above-mentioned amplification molecular markers using primers SEQ ID NO.2 and SEQ ID NO.3.

[0012] The amplification system is 25 μL, and its components include: 12.5 μL 2×Rapid Taq Master Mix, 1 μL DNA template, 10 primers (left and right) mol / L 1 μL each, 9.5 μL dd H2O.

[0013] The amplification program was as follows: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 60 s, for a total of 35 cycles; 72℃ for 5 min.

[0014] The fifth objective of this invention is to provide a method for selecting maize varieties with shorter tassel main axis length: Extracting genomic DNA from maize varieties; Using maize genomic DNA as a template, PCR amplification was performed using the left primer F and the right primer R to obtain the amplification product; The amplified products were sequenced and identified.

[0015] Furthermore, if the identification results show that a maize variety simultaneously has a 19 bp deletion (SEQ ID NO.4) at 151,876,914-151,876,932 bp, a 40 bp insertion (SEQ ID NO.5) at 151,876,933 bp, and an 8 bp insertion (SEQ ID NO.6) at 151,877,050 bp on chromosome 7 of the reference genome (Zm-B73-REFERENCE-NAM-5.0), its tassel main axis is shorter; The main axis of the tassel of this variety is relatively long when there is a 4 bp deletion (SEQ ID NO.7) at 151,877,268-151,877,271 bp, a 5 bp deletion (SEQ ID NO.8) at 151,877,386-151,877,390 bp, a 6 bp insertion (SEQ ID NO.9) at 151,877,438 bp, a 4 bp insertion (SEQ ID NO.10) at 151,877,587 bp, and a 7 bp deletion (SEQ ID NO.11) at 151,877,634-151,877,640 bp.

[0016] Furthermore, the nucleotide sequences of SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11 are as follows: SEQ ID NO.4: TGACATGGCCCCCACCCTT; SEQ ID NO.5: GGCCGGCGGCCGTGGCGCCACGTGACCTGACAAGGCCCAA; SEQ ID NO.6: GCCCGCCG; SEQ ID NO.7: TGAG; SEQ ID NO.8: GACCT; SEQ ID NO.9: CCAGCA; SEQ ID NO.10: CATA; SEQ ID NO.11: ACGGACC.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a highly efficient molecular-assisted selection method for breeding maize varieties with shorter tassel main axes. This technology significantly improves breeding selection efficiency, provides a powerful tool for breeding high-yielding and density-tolerant maize varieties, and has significant application value and promising prospects for achieving high and stable maize yields. Attached Figure Description

[0018] Figure 1 The image shows the amplified molecular marker bands of maize varieties MY73, YD819, Kangnongyu 8009, Yudan 827, Qiule 999, Xianyu 1466, Xianyu 335, and Xiuqing 835 in Example 2 of this invention.

[0019] Figure 2 This document describes the sequence alignment differences and tassel length of maize varieties MY73, YD819, Kangnongyu 8009, Yudan 827, Qiule 999, Xianyu 1466, Xianyu 335, and Xiuqing 835 in Example 2 of this invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to its embodiments; it should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0021] Example 1: Determination and development of molecular markers in this invention.

[0022] This invention used 368 major maize varieties with broad genetic diversity as materials, and performed genome-wide genotyping using the Maize 48KBead Chip chip. Genome-wide association analysis (GWAS) was then conducted in conjunction with field data on tassel axis length phenotypes. The results identified one tightly linked genetic locus significantly associated with tassel axis length. By integrating public genome databases, this locus was found to be associated with… ZmTHX48 Related.

[0023] Based on the above findings, we targeted candidate genes ZmTHX48 Specific molecular markers were developed for the promoter and exon regions of the target genome, and the nucleotide sequence of these markers is shown in SEQ ID NO.1.

[0024] SEQ ID NO.1: Primers for amplifying the aforementioned molecular marker were developed, and the nucleotide sequences of the left primer F (as shown in SEQ ID NO.2) and the right primer R (as shown in SEQ ID NO.3) are as follows: Left primer F (SEQ ID NO.2): TACTGCTACTGCATCGGTCG; Right primer R (SEQ ID NO.3): GCGTTTCCGTTCCAACCCC.

[0025] Example 2: In this example, the molecular markers were verified using maize varieties MY73, YD819, Kangnongyu 8009, Yudan 827, Qiule 999, Xianyu 1466, Xianyu 335, and Xiuqing 835.

[0026] Primers were used to amplify and sequence maize varieties MY73, YD819, Kangnongyu 8009, Yudan 827, Qiule 999, Xianyu 1466, Xianyu 335, and Xiuqing 835. Comparison of the sequences of each variety revealed the following: Maize varieties with shorter tassel main axis (MY73, YD819, Kangnongyu 8009, and Yudan 827) have a 19 bp deletion (SEQ ID NO.4) at 151,876,914-151,876,932 bp, a 40 bp insertion (SEQ ID NO.5) at 151,876,933 bp, and an 8 bp insertion (SEQ ID NO.6) at 151,877,050 bp on chromosome 7 of the reference genome (Zm-B73-REFERENCE-NAM-5.0). Maize varieties with relatively long tassel main axes (Qiule 999, Xianyu 1466, Xianyu 335, and Xiuqing 835) have the following deletions on chromosome 7 of the reference genome (Zm-B73-REFERENCE-NAM-5.0): a 4 bp deletion (SEQ ID NO.7) at 151,877,268-151,877,271 bp, a 5 bp deletion (SEQ ID NO.8) at 151,877,386-151,877,390 bp, a 6 bp insertion (SEQ ID NO.9) at 151,877,438 bp, a 4 bp insertion (SEQ ID NO.10) at 151,877,587 bp, and a 7 bp deletion (SEQ ID NO.11) at 151,877,634-151,877,640 bp.

[0027] The nucleotide sequences of the fragments (SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11) are as follows: SEQ ID NO.4: TGACATGGCCCCCACCCTT; SEQ ID NO.5: GGCCGGCGGCCGTGGCGCCACGTGACCTGACAAGGCCCAA; SEQ ID NO.6: GCCCGCCG; SEQ ID NO.7: TGAG; SEQ ID NO.8: GACCT; SEQ ID NO.9: CCAGCA; SEQ ID NO.10: CATA; SEQ ID NO.11: ACGGACC.

[0028] Figure 1 This document shows the amplified molecular marker banding patterns of maize varieties MY73, YD819, Kangnongyu 8009, Yudan 827, Qiule 999, Xianyu 1466, Xianyu 335, and Xiuqing 835 in this embodiment. Based on... Figure 1 It can be seen that when primers were used to amplify maize varieties MY73, YD819, Kangnongyu 8009, Yudan 827, Qiule 999, Xianyu 1466, Xianyu 335, and Xiuqing 835, the bands were clearly visible and their size matched the target bands of the molecular markers.

[0029] Figure 2 This document describes the sequence alignment differences and tassel length characteristics of maize varieties MY73, YD819, Kangnongyu 8009, Yudan 827, Qiule 999, Xianyu 1466, Xianyu 335, and Xiuqing 835 in this embodiment. Based on... Figure 2It was found that maize varieties with shorter tassel main axis length (28.66 cm ~ 31.16 cm) (MY73, YD819, Kangnongyu 8009, and Yudan 827) had a 19 bp deletion (SEQ ID NO.4) at 151,876,914-151,876,932 bp, a 40 bp insertion (SEQ ID NO.5) at 151,876,933 bp, and an 8 bp insertion (SEQ ID NO.6) on chromosome 7 of the reference genome (Zm-B73-REFERENCE-NAM-5.0); while maize varieties with longer tassel main axis length (41.37 cm ~ 44.06 cm) had a 19 bp deletion (SEQ ID NO.4) at 151,876,914-151,876,932 bp, and a 40 bp insertion (SEQ ID NO.5) at 151,876,933 bp, and a 40 bp insertion (SEQ ID NO.6) at 151,877,050 bp. Maize varieties (Qiule 999, Xianyu 1466, Xianyu 335, and Xiuqing 835) have the following deletions on chromosome 7 of the reference genome (Zm-B73-REFERENCE-NAM-5.0): a 4 bp deletion (SEQ ID NO.7) at positions 151,877,268-151,877,271 bp, a 5 bp deletion (SEQ ID NO.8) at positions 151,877,386-151,877,390 bp, a 6 bp insertion (SEQ ID NO.9) at position 151,877,438 bp, a 4 bp insertion (SEQ ID NO.10) at position 151,877,587 bp, and a 7 bp deletion (SEQ ID NO.11) at positions 151,877,634-151,877,640 bp. Yellow triangles indicate insertions, and white spaces indicate deletions.

[0030] Example 3: Reference parameters for the process of amplifying molecular markers using primers of the present invention.

[0031] 1) DNA extraction: Tiangen Biotech's new plant genomic DNA extraction kit (centrifuge column type), after DNA concentration was measured, diluted to 15%. g / mL.

[0032] 2) 25 L reaction system (as shown in Table 1).

[0033]

[0034] 3) PCR amplification procedure (as shown in Table 2).

[0035]

[0036] 4) Electrophoresis: 1% agarose gel; 1×TAE buffer; electrophoresis at 120V and 150mA for 15 minutes.

[0037] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A molecular marker for regulating tassel primary axis length in maize, comprising, This molecular marker is located on chromosome 7 of the maize reference genome (Zm-B73-REFERENCE-NAM-5.0), with a 19 bp deletion at 151,876,914-151,876,932 bp, a 40 bp insertion at 151,876,933 bp, and an 8 bp insertion at 151,877,050 bp. Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The primers for amplifying the molecular marker of claim 1, characterized in that, The nucleotide sequences of the left primer F and the right primer R are as follows: Left primer F (SEQ ID NO.2): TACTGCTACTGCATCGGTCG; Right primer R (SEQ ID NO.3): GCGTTTCCGTTCCAACCCC.

3. The application of the molecular marker of claim 1 or the primer for amplifying the molecular marker of claim 2 in the breeding of maize varieties with shorter tassel main axis.

4. A method for breeding maize varieties with shorter tassel main axis, characterized in that, Includes the following steps: Extracting genomic DNA from maize varieties; Using maize genomic DNA as a template, PCR amplification was performed using the left primer F and the right primer R to obtain the amplification product; The amplification products were sequenced and identified. If the identification results show that the maize carries the characteristic sequence of the molecular marker described in claim 1, then the maize is a variety with a shorter main axis of tassel.

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