Molecular Markers Closely Linked to the Major QTL for Ear Diameter in Maize and Their Applications

Abstract

The present invention discloses a molecular marker closely linked to a major ear diameter QTL in maize, as well as its primers and applications, belonging to the field of biotechnology. The molecular marker is located on chromosome 5 of maize and includes the molecular marker chr5-30 and the molecular marker M3. The primer sequences for amplifying the molecular marker chr5-30 are: chr5-30-L: 5'-AAACAAATCTATGCGCCCAC-3'; chr5-30-R: 5'-AATGGACCGGTTCTCCTAGC-3'; for amplifying the molecular marker M3: M3-L: 5'-TTCCTACCTGCGGTACTTGC-3'; M3-R: 5'-GGTGGTGCCTGCTACAGAGT-3'. The molecular marker provided by the present invention is closely linked to the major ear diameter QTL in maize, and it and its primers can be applied to molecular marker-assisted breeding for ear diameter, screening of germplasm resources for ear diameter, and genetic improvement of maize ear diameter.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a molecular marker closely linked to a major QTL for maize ear diameter and its application. Background Art

[0002] Maize is a very important food crop and cash crop in China, and it is also the crop with the highest total world output. At the same time, it is an important renewable industrial raw material. With the rapid development of social economy and the urgent needs of the economic market, continuously increasing maize yield is necessary to meet the needs of rapid economic development and the market demand for maize-derived foods, feeds, and biofuels. Maize yield per mu is mainly determined by the number of ears per mu and the grain weight per ear. Among them, the grain weight per ear can be further decomposed into three elements: ear length (number of grains per row), ear diameter (number of rows of ears), and 100-grain weight. As one of the first breeding objectives considered by breeders, screening and identifying germplasm resources with large ear diameter is an important way to cultivate new high-yield maize varieties.

[0003] Maize ear diameter is a typical quantitative trait and is easily affected by environmental conditions. There are problems such as long breeding cycles and low efficiency in selecting germplasm resources with large ear diameter through traditional breeding methods. Some studies have tried various technical means to mine and clone related genes. But so far, only a very small number of maize ear diameter QTLs have been finely mapped or cloned. Therefore, developing molecular markers for cultivating maize with large ear diameter has become a key task in the genetic improvement of maize ear diameter.

[0004] QTL mapping is an important step in isolating target genes and their closely linked molecular markers. An appropriate mapping population is crucial for the speed and accuracy of QTL mapping. A single segment substitution line refers to a line in which, except for the target segment, the rest of the chromosome is identical to the recipient. Such materials have a single genetic background, which can eliminate the influence of the genetic background in QTL analysis, improving the accuracy of mapping; at the same time, it avoids the interference of epistatic interactions, enabling minor QTLs to be detected and enhancing the QTL discrimination ability; by comparing different single segment substitution lines, the target QTL region can be directly identified, decomposing multiple loci of quantitative traits into multiple single Mendelian factors, simplifying the research process of quantitative traits. Therefore, single segment substitution lines have been widely used in QTL cloning and functional analysis.

[0005] At present, although single segment substitution lines have been used to map maize ear diameter genes, the obtained mapping intervals are relatively large, and it is difficult to apply the molecular markers in production. Therefore, it is necessary to develop a molecular marker closely linked to the target gene for genetic improvement of maize ear diameter. Summary of the Invention

[0006] The object of the present invention is to provide a molecular marker closely linked to the major QTL for maize ear diameter and its application. This molecular marker is closely linked to the major QTL for maize ear diameter and can be used for molecular marker-assisted breeding of maize ear diameter.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A molecular marker closely linked to the major QTL for maize ear diameter, the molecular marker is located on chromosome 5 of maize, and is the molecular marker chr5-30 and the molecular marker M3.

[0009] Furthermore, the specific position of the molecular marker chr5-30 is chr5: 84399694-84400040, and the specific position of the molecular marker M3 is chr5: 85072289-85072443. The physical position of the present invention refers to the V5 version.

[0010] Primers for the molecular marker closely linked to the major QTL for maize ear diameter, the primer sequences for amplifying the molecular marker chr5-30 are:

[0011] chr5-30-L: 5’-AAACAAATCTATGCGCCCAC-3’ (Sequence 1);

[0012] chr5-30-R: 5’-AATGGACCGGTTCTCCTAGC-3’ (Sequence 2);

[0013] For amplifying the molecular marker M3:

[0014] M3-L: 5’-TTCCTACCTGCGGTACTTGC-3’ (Sequence 3);

[0015] M3-R: 5’-GGTGGTGCCTGCTACAGAGT-3’ (Sequence 4).

[0016] Application of the molecular marker closely linked to the major QTL for maize ear diameter in the genetic improvement of maize ear diameter.

[0017] Furthermore, the method for identifying the maize ear diameter trait in the genetic improvement of maize ear diameter includes the following steps:

[0018] Extract the genomic DNA of maize leaves;

[0019] Using the genomic DNA of maize leaves as a template, perform PCR amplification respectively with the primers chr5-30-L / chr5-30-R, M3-L / M3-R;

[0020] Identification of PCR amplification results by agarose gel electrophoresis: When the primers used are chr5-30-L / chr5-30-R, if the molecular marker chr5-30 is detected as the upper band, it indicates that the ear diameter of the sample to be tested is relatively thick; if the molecular marker chr5-30 is detected as the lower band, it indicates that the ear diameter of the sample to be tested is relatively thin. When the primers used are M3-L and M3-R, if the molecular marker M3 is detected as the upper band, it indicates that the ear diameter of the sample to be tested is relatively thick; if the molecular marker M3 is detected as the lower band, it indicates that the ear diameter of the sample to be tested is relatively thin.

[0021] Furthermore, the PCR amplification system is 10 μL, and the components include: 2 μL of DNA, 0.5 μL of each of the left and right primers, 5 μL of 2×Taq Master Mix, and 2 μL of dd H2O.

[0022] Furthermore, the Touchdown PCR amplification program is used: 95°C for 3 min; 95°C for 30 s, 65°C for 30 s, with a 1°C decrease in each cycle, 72°C for 30 s, for a total of 9 cycles; 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, for a total of 29 cycles; 72°C for 5 min.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] The molecular marker of the present invention that is closely linked to the major ear diameter QTL in maize is closely linked to the major ear diameter QTL in maize, and can be applied to molecular marker-assisted breeding for ear diameter, screening of germplasm resources for ear diameter, and genetic improvement of maize ear diameter. Description of the Drawings

[0025] Figure 1 It is a comparison diagram of ear diameter between single segment substitution line SSSL1272 and Xu 178;

[0026] Figure 2 It is a distribution diagram of 3 pairs of differential markers on chromosome 5;

[0027] Figure 3 It is the target gene within 1.08 M on chromosome 5;

[0028] Figure 4 It is a linkage differential marker diagram. Detailed Embodiments

[0029] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings.

[0030] In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0031] Example 1

[0032] This example provides a process for locating a major QTL for maize ear diameter, as follows:

[0033] (1) Construction of a population of maize single segment substitution lines:

[0034] In the early stage of the laboratory, the single segment substitution line was constructed by combining multiple generations of backcrossing and selfing with SSR molecular marker-assisted selection using three donor parents of the corn inbred line ensemble and Xu 178 as the recipient parent. Through investigation and identification at six test sites in Xinxiang, Junxian and Xuchang over two years, and through investigation of the ear traits of the single segment substitution line and Xu 178, it was found that the ear diameter of the single segment substitution line SSSL1272 was significantly different from that of Xu 178. The results are shown in Figure 1 This indicates that there is a major QTL controlling ear diameter in this single segment substitution line.

[0035] (2) Preliminary positioning of the main QTL qED5 for maize ear diameter:

[0036] 2.1 Construction of positioning population: Single segment substitution lines SSSL1272 and Xu 178 were planted in the experimental bases in Yuanyang, Henan and Sanya, Hainan, with SSSL1272 as the female parent and Xu 178 as the male parent, and the hybrid group was F1. F1 and Xu 178 were planted in the Hainan experimental base, and a new BC1F1 population was constructed with F1 as the female parent and Xu 178 as the male parent.

[0037] 2.2 Identification of ear thickness phenotype: After harvest in October 2019, the ear traits of individual plants in the BC1F1 population were investigated, and the extreme phenotypes of extremely thick and extremely thin were selected from the backcross first-generation population. The self-pollinated progeny BC1F2 of individual plants were planted and identified in the experimental bases in Sanya, Hainan and Yuanyang, Henan in December 2019 and June 2020, respectively, with 1.3 replicates set up. In March 2020 and October 2020, the ear traits of the harvested individual plants were measured using a seed testing instrument.

[0038] In December 2020, the BC1F2 population was used to screen new exchange plants. In June 2021 and December 2021, the self-pollinated progeny of the exchange plants were selected and planted in Yuanyang, Henan and Hainan for phenotype identification.

[0039] 2.3 DNA extraction and molecular marker development

[0040] The genomic DNA of maize leaves was extracted by SLS method and stored in a -20℃ refrigerator for later use.

[0041] From the IBM 2008 Neighbors Frame2 molecular marker reference map in the MaizeGDB (http: / / www.maizegdb.org / ) database, 1000 pairs of SSR primers covering the whole maize genome were selected.

[0042] InDel marker development: By aligning the genomic sequences of Xu 178 and Zong 3, sites with sequence differences of more than 5 bases between the two materials were selected, and primers were designed using Primer3.0. The primer length was between 18 - 24 bp, and the specific sequences are shown in Table 1.

[0043] 2.4 PCR procedure and genotyping analysis of amplification products

[0044] The components of the PCR amplification system (10 μL) include: 2 μL of DNA, 1 μL of primer (0.5 μL for each of the left and right primers), 5 μL of 2×Taq Master Mix (Novizan), and 2 μL of dd H2O. The Touchdown PCR amplification procedure was used: 95°C for 3 min; 95°C for 30 s, 65°C for 30 s (with a 1°C decrease in each cycle), 72°C for 30 s, for a total of 9 cycles; 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, for a total of 29 cycles; 72°C for 5 min. The genotypes of the PCR amplification products were analyzed by polyacrylamide gel electrophoresis and agarose gel electrophoresis.

[0045] 2.5 Preliminary mapping of major QTL for ear diameter in maize

[0046] A total of 1000 SSR markers evenly covering the 10 chromosomes of maize were used to screen for polymorphic markers in the whole genomes of the two parents, Xu 178 and SSSL1272. Ten individual plants with significantly thick ears and ten individual plants with significantly thin ears were selected from the BC1F1 segregating population, and their DNAs were mixed equally to construct a dominant pool and a recessive pool. Three pairs of differential markers were screened and were located on chromosome 5. The results are shown in Figure 2 . From left to right in the figure are SSSL1272, Xu 178, the dominant pool, and the recessive pool. Another 250 pairs of SSR and Indel markers were developed using the sequences of inbred lines B73 and W22. By using the BSA pooling method, 6 more pairs of differential markers were screened. The primer information for the molecular markers is shown in Table 1.

[0047] Table 1 Primer information for molecular markers

[0048]

[0049] By identifying the BC1F1 offspring in Hainan and Yuanyang in December 2019 and June 2020, each offspring of the exchanged individual plants was randomly self-crossed in one row, and the seeds were harvested and examined for traits to identify the phenotypes of the BC1F1. Seventy individual plants with phenotypes were selected. Finally, the target gene was mapped between two markers, umc1815 and 5.04 - InDel - 19, on chromosome 5, and the target region was locked within a range of 1.08 M. The results are shown in Figure 3 . Figure 3The white color represents the fragment from Xu 178, and the black color represents the heterozygous fragment of Xu 178 and SSSLqED5. The bar chart on the right represents the average ear diameter of the selfed progeny of the recombinant single plants, and the significant difference P is obtained by t-test with Xu 178. The black dotted line represents the differential marker inside.

[0050] (3) Fine mapping of the major QTL qED5 for maize ear diameter:

[0051] 3.1 Molecular marker development: To further narrow down the mapping range and achieve the fine mapping of the major QTL for ear diameter, according to the differences between the sequences of maize inbred line B73 and Xu 178, within the initially mapped interval of 1.08 M, Indel and SSR markers were further developed, and differential markers were screened with Xu 178 and SSSLqED5. Six differential markers were found, and the specific sequences are shown in Table 2.

[0052] Table 2 Primer information of molecular markers

[0053]

[0054]

[0055] 3.2 Construction of the mapping population: Single plants with heterozygous target segments were selected from the BC1F1 population for selfing to construct the BC1F2 population.

[0056] 3.3 Identification of ear diameter phenotypes: In 2021, two linked markers, umc1815 and 5.04-InDel-19, were used to screen a population of 20,000 BC1F2 seeds. The selected recombinant single plants were planted in the late summer in Yuanyang in 2021, and samples were taken to extract DNA. The recombinant single plants within this segment were screened using the markers umc1815 and 5.04-InDel-19. These homozygous recombinant single plants were identified with the newly developed molecular markers, and 11 new exchange types were found. After harvesting, the phenotypes were identified by planting in Yuanyang and Hainan in 2022.

[0057] 3.4 PCR program and genotype analysis of amplification products: The components of the PCR amplification system (10 μL) include: 2 μL of DNA, 1 μL of primer (0.5 μL for each of the left and right primers), 5 μL of 2×Taq Master Mix (Novizan), and 2 μL of ddH2O. The Touchdown PCR amplification program was used: 95°C for 3 min; 95°C for 30 s, 65°C for 30 s (decreasing 1°C for each cycle), 72°C for 30 s, for a total of 9 cycles; 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, for a total of 29 cycles; 72°C for 5 min. The genotypes of the PCR amplification products were analyzed by polyacrylamide gel electrophoresis and agarose gel electrophoresis.

[0058] 3.5 Fine mapping of the major QTL for maize ear diameter

[0059] The ear traits of the harvested materials were identified using a maize seed analyzer. After analyzing the seed analysis data, by combining linkage differential markers and the results of field trait identification, the target gene was mapped between two linkage differential markers, chr5-30 and MC-10, and the physical distance between the two was 673 kb. The results are shown in Figure 4 . The white box represents the genomic fragment from Xu 178, and the black box represents the genomic fragment of SSSL1272. The significant difference P was obtained by performing a t-test with Xu 178.

[0060] Example 2

[0061] The molecular markers closely linked to the major QTL for maize ear diameter obtained in Example 1 can be applied in the genetic improvement of maize ear diameter. The method for identifying the maize ear diameter trait in the process of maize ear diameter molecular marker-assisted breeding includes the following steps:

[0062] Extract the genomic DNA of maize leaves;

[0063] Using the genomic DNA of maize leaves as a template, perform PCR amplification using primers chr5-30-L / chr5-30-R and M3-L / M3-R respectively;

[0064] Identify the PCR amplification results by agarose gel electrophoresis: When the primers used are chr5-30-L / chr5-30-R, when it is detected that the molecular marker chr5-30 is the upper band, it indicates that the ear diameter of the sample to be tested is thicker; when it is detected that the molecular marker chr5-30 is the lower band, it indicates that the ear diameter of the sample to be tested is thinner; when the primers used are M3-L and M3-R, when it is detected that the molecular marker M3 is the upper band, it indicates that the ear diameter of the sample to be tested is thicker; when it is detected that the molecular marker M3 is the lower band, it indicates that the ear diameter of the sample to be tested is thinner.

[0065] Among them, the PCR amplification system is 10 μL, and the components include: 2 μL of DNA, 0.5 μL of each of the left and right primers, 5 μL of 2×Taq Master Mix, and 2 μL of ddH2O. Use the Touchdown PCR amplification program: 95°C for 3 min; 95°C for 30 s, 65°C for 30 s, with a 1°C drop in each cycle, 72°C for 30 s, for a total of 9 cycles; 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, for a total of 29 cycles; 72°C for 5 min.

[0066] It should be noted that when the present invention involves numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the adopted step methods are the same as those in the embodiments, in order to prevent repetition, the present invention describes the preferred embodiments. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0067] Obviously, those skilled in the art can 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 of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. Application of primers for amplifying molecular markers tightly linked to the major QTL for maize ear diameter in genetic improvement of maize ear diameter, characterized in that, The molecular markers are located on chromosome 5 of maize, and are molecular marker chr5-30 and molecular marker M3; The primers for amplifying the molecular markers are: The primer sequences for amplifying the molecular marker chr5-30 are: chr5-30-L: 5’-AAACAAATCTATGCGCCCAC-3’; chr5-30-R: 5’-AATGGACCGGTTCTCCTAGC-3’; The primer sequences for amplifying the molecular marker M3 are: M3-L: 5’-TTCCTACCTGCGGTACTTGC-3’; M3-R: 5’-GGTGGTGCCTGCTACAGAGT-3’; The method for identifying the ear diameter trait of maize in the genetic improvement of maize ear diameter includes the following steps: Extract the genomic DNA of maize leaves; Using the genomic DNA of maize leaves as a template, perform PCR amplification respectively with the primers chr5-30-L / chr5-30-R, M3-L / M3-R.

2. Application of primers for amplifying molecular markers tightly linked to the major QTL for maize ear diameter in genetic improvement of maize ear diameter according to claim 1, characterized in that, The amplification system for performing PCR amplification respectively with the primers chr5-30-L / chr5-30-R, M3-L / M3-R is 10 μL, and the components include: 2 μL of DNA, 0.5 μL of each of the left and right primers, 5 μL of 2×TaqMaster Mix and 2 μL of dd H2O.

3. Application of primers for amplifying molecular markers tightly linked to the major QTL for maize ear diameter in genetic improvement of maize ear diameter according to claim 1, characterized in that, Use the Touchdown PCR amplification program: 95℃ for 3 min; 95℃ for 30 s, 65℃ for 30 s, with a 1℃ drop for each cycle, 72℃ for 30 s, for a total of 9 cycles; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, for a total of 29 cycles; 72℃ for 5 min.

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