A molecular marker for identifying the trait of multiple axillary branches in millet and its application

Abstract

The present invention belongs to the field of variety identification, and specifically relates to a molecular marker for identifying the leaf axillary branching trait of millet and its application. The present invention identifies the leaf axillary branching genetic regulatory sites and target genes of millet vegetative branching trait by forward genetics and develops tightly linked markers thereof, which is of great significance for molecular assisted selection and breeding of forage millet varieties.

Description

Technical Field

[0001] The invention belongs to the field of variety identification, and in particular relates to a molecular marker for identifying millet leaf axillary branching traits and an application thereof. Background Art

[0002] Millet is one of the earliest domesticated cereal crops and has a cultivation history of more than 8,000 years in China. It is a specialty crop for both grain and feed in my country's arid and semi-arid areas. It is drought-resistant, barren-resistant and has high nutritional value. Modern cultivated varieties are mainly grain varieties, which are characterized by no vegetative branches or a few vegetative branches - leaf axillary branches, thick stems, and similar grain-to-grass ratios, making them unsuitable for feeding. Forage varieties should have plant characteristics such as higher biomass, lower grain-to-grass ratio, thinner stems, and lush leaves, all of which are closely related to vegetative branching. Compared with major crops such as rice, corn, and wheat, there are few reports on the mining of millet vegetative branching genetic loci and genes. The major effect genes MOC1, TAD1, LAX1, BA1, TB1, etc. that mainly regulate the vegetative branching traits of cereal crops have not been reported in millet. Therefore, it is of great significance to identify the vegetative branching traits of millet - tillering, leaf axillary branching genetic regulatory loci, target genes, and develop their tightly linked markers through forward genetics, which is of great significance for molecular-assisted selection and breeding of forage millet varieties. Summary of the invention

[0003] To solve the above problems, the present invention provides a primer set of InDel marker GSA7.548 for identifying the multi-leaf axillary branching trait of millet, including a forward primer as shown in SEQ ID NO.1 in the sequence listing; and a reverse primer as shown in SEQ ID NO.2 in the sequence listing.

[0004] The present invention also provides a kit, comprising the above primer set.

[0005] Furthermore, the above-mentioned kit also includes reagents required for PCR reaction and / or electrophoresis.

[0006] The present invention also provides the application of the primer set in identifying the multi-leaf axillary branching trait of millet.

[0007] The invention also provides application of the kit in identifying the multi-leaf axillary branching trait of millet.

[0008] The present invention also provides a method for identifying the multi-leaf axillary branching trait of millet, comprising the following steps:

[0009] (1) Leaf collection at the millet seedling stage: When millet is at the 3-5 leaf stage, fresh leaves of the test germplasm materials are collected and stored at -80°C for DNA extraction;

[0010] (2) Extracting DNA from samples;

[0011] (3) PCR reaction: Use a 96-well PCR plate, configure the PCR reaction system, and perform PCR amplification reaction on a thermal cycler;

[0012] (4) Polyacrylamide gel electrophoresis of PCR products;

[0013] (5) Genotype reading: Based on the band pattern in the film in step (4), the presence of a band at 223 bp indicates that the sample genotype is a multi-leaf axillary branching trait.

[0014] Furthermore, the reaction procedure is: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, a total of 35 cycles; extension at 72°C for 10 minutes.

[0015] The present invention also provides an InDel marker GSA7.548 for identifying the multi-leaf axillary branching trait of millet, which is amplified by the above primer set.

[0016] The present invention has the following beneficial effects:

[0017] The technology of the present invention is aimed at the actual needs of molecular assisted selection breeding of forage millet. It targets the nutritional branching trait closely related to millet biomass, adopts forward genetics, and uses interspecific populations to identify the main genetic regulatory sites of the millet nutritional branching trait - axillary branching, providing technical support for the development of its molecular markers and the rapid identification of multi-axillary branching germplasm and subsequent breeding materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 GSA7.548 marker sequence primers and physical location;

[0020] Figure 2 Genotypic characteristics of GSA7.548, BB is a genotype with multiple axillary branches;

[0021] Figure 3The results of phenotypic variance analysis of the axillary branching traits corresponding to all families of the genotypes AA (Yugu No. 1 genotype) and BB (Setaria genotype) produced by GSA7.548 marker in the F2, F3, and F4 populations of Yugu No. 1 × Setaria H1. A and B indicate that the genotypes have extremely significant differences in phenotypes in the F2, F3, and F4 populations (p < 0.01). The phenotype corresponding to the AA genotype is the offspring material with few axillary branches; the phenotype corresponding to the BB genotype is the offspring material with many axillary branches. DETAILED DESCRIPTION

[0022] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] This patent uses the non-nutritional branching millet variety Yugu No. 1 (introduced from Anyang Academy of Agricultural Sciences) and the multi-nutritional branching millet wild ancestor Setaria viridis H1 (from Gansu Academy of Agricultural Sciences) to construct F2 and F 2:3 、F 2:4 The pool b1 was constructed by selecting 30 families with no tillering and no axillary branches in the F2 population, and the pool b2 was constructed by selecting 30 families with many tillers and many axillary branches. BSA-seq was used to calculate the ΔSNP-Index between the pools, and the associated loci regulating vegetative branching were analyzed. Two associated loci were identified on chromosome 5 of millet, with physical intervals of 10892194-11048639bp and 31457185-41949497bp, respectively. One associated locus of 4983165--8390969bp was identified on chromosome 9. In order to distinguish the genetic regulatory loci of the two vegetative branching traits - tillering and axillary branches, the F 2:3 38 families with no tillers and no axillary branches were selected from the population to construct the mixed pool B1, 38 families with many tillers and many axillary branches were constructed to construct the mixed pool B2, and 38 families with many tillers and no axillary branches were constructed to construct the mixed pool B3. BSA-seq was used to calculate the ΔSNP-Index between the B1 and B3 pools (the two pools only differed in the axillary branching trait). The results showed that two loci regulating the axillary branching trait were identified in the intervals of 38452384-40147288bp on chromosome 2 and 4350040-10448047bp on chromosome 9 of millet. The associated locus on chromosome 9 overlapped with the associated locus 4983165-8390969bp identified in the F2 population, indicating that this locus is the key locus regulating the axillary branching trait of millet and was named qAB9.1.

[0028] To further narrow the qAB9.1 physical interval, 24 SSR markers and 38 InDel markers were developed within the qAB9.1 interval, using 559 F 2:4 The recombinant plant finely located qAB9.1 to the interval of 7465289-7986584bp on chromosome 9. This physical interval contains 9 InDel markers, among which InDel marker GSA7.548 is an insertion / deletion fragment of gene Seita.9G1217005'UTR, which lacks CTGCACCA 8 bases in Yugu No. 1 and inserts CTGCACCA 8 bases in Setaria H1, and its conservation is strong. In addition, qAB9.1 is highly conserved in F2 and F 2:3 、F 2:4 The LOD values ​​were 10.1, 9.7, and 4.2, respectively, which was a major effect site for the vegetative branching-axillary branching trait.

[0029] Example 1

[0030] 1. Leaf collection at the millet seedling stage: When millet is at the 3-5 leaf stage, collect fresh leaves of the test germplasm materials, bring them back to the laboratory, and store them at -80℃ for DNA extraction.

[0031] 2. Extraction of millet DNA: The following method is used for extraction:

[0032] 2.1 Prepare two sets of 2ml and 1.5ml centrifuge tubes respectively and number them for use;

[0033] 2.2 Take about 1 g of fresh leaves from step 1 and put them into a 2 ml centrifuge tube with 5 mm steel balls, freeze them quickly with liquid nitrogen, grind them into powder using a sample group tissue grinder, add 1 ml of preheated (65°C) lysis buffer (Table 1), mix quickly, put them in a 65°C water bath, and bathe them for 40-60 minutes. Invert and mix several times during the water bath;

[0034] Table 1 Lysis buffer configuration

[0035]

[0036] 2.3 After completing step 2.2, add 1 ml of chloroform:isoamyl alcohol (24:1), shake several times to emulsify, let stand at room temperature for 10 min, and centrifuge at 10000 rpm for 10 min (4°C);

[0037] 2.4 After centrifugation, take 700ul of supernatant into the corresponding 1.5ml centrifuge tube, add an equal volume of -20℃ pre-cooled isopropanol, gently invert several times, and centrifuge at 10000rpm for 1min to allow the DNA to precipitate to the bottom of the tube;

[0038] 2.5 Pour off the supernatant in the centrifuge tube, add 500ul 75% ethanol, rinse twice, then rinse once with anhydrous ethanol, and let the white DNA air dry in the tube;

[0039] 2.6 Add 200ul TE solution and place in a -20℃ refrigerator for later use.

[0040] 3. GSA7.548 mark (with Figure 1 ) PCR reaction system: Referring to Table 2, select 96 empty PCR plates, configure the PCR reaction system, and perform PCR amplification reaction on a thermal cycler. The reaction procedure is: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 1 minute, a total of 35 cycles; 72°C extension for 10 minutes.

[0041] Table 2 PCR reaction system

[0042]

[0043] 4. Polyacrylamide gel electrophoresis of PCR products labeled with GSA7.548 was performed using a DYY-6C electrophoresis instrument produced by Beijing Liuyi Factory and a DYCZ-28A electrophoresis tank.

[0044] 4.1 Use a pipette to add 2.0ul of loading buffer (Table 3) to the PCR product obtained in step 3 and let it stand for use;

[0045] Table 3 Loading buffer configuration

[0046]

[0047] 4.2 Install the electrophoresis tank: Put the cleaned glass plate into the rubber strip, seal the gap between the glass plate and the rubber strip with 1% agarose gel dissolved by heating, cool for 5 minutes, gently pick up the rubber strip and glass plate with both hands and put them into the glass tank, and fix them evenly with screws.

[0048] 4.3 Gel preparation: Prepare 30% polyacrylamide gel (Table 4), mix well and quickly pour into the glass slit, gently insert the comb, and invert the electrophoresis tank for about 40 minutes until the gel is completely solidified.

[0049] Table 4 30% acrylamide gel solution configuration

[0050]

[0051] 4.4 Loading PCR products: Pour enough TBE buffer (Table 5) into the electrophoresis tank, gently remove the comb, and use a 10ul pipette to draw 2ul of the sample in step 4.1 and carefully add it to the comb hole.

[0052] Table 5 TBE buffer configuration

[0053]

[0054] 4.5 Electrophoresis: Run at 350 V for about 40 min, and terminate when the bromophenol blue is 1 cm away from the bottom of the electrophoresis tank.

[0055] 4.6 Silver staining: Take out the gel from the electrophoresis tank and glass plate, pour in 500 ml of silver staining solution (Table 6), place on a shaker and shake gently for 10 minutes.

[0056] Table 6 Silver stain solution configuration

[0057]

[0058] 4.7 Rinse: Discard the silver staining solution, pour in an appropriate amount of deionized water, and gently shake several times to ensure even rinsing.

[0059] 4.8 Color development: discard the rinse solution, add the color development solution (Table 7), and gently shake on a shaker until clear bands appear.

[0060] Table 7 Color development solution configuration

[0061]

[0062] 4.9 Washing the gel: discard the color developing solution and rinse the gel with tap water several times to remove the formaldehyde smell.

[0063] 4.10 Take photos: Spread the gel from step 4.9 on a light box and take photos.

[0064] 5. Genotype reading: According to the band pattern on the film in step 4.10, refer to the attached Figure 2 Read the material genotype, AA is the germplasm genotype with few axillary branches, and BB is the germplasm genotype with many axillary branches.

[0065] Example 2

[0066] 1. For the three generation populations of Yugu No. 1×Setaria officinalis H1 F2, F3, and F4, 897, 890, and 1685 materials, respectively, fresh leaves were collected at the booting stage in 2019, 2020, and 2021, and DNA was extracted from each material using step 2 in 4.2.2;

[0067] 2. Amplify the target fragment using step 3 in 4.2.2;

[0068] 3. Perform polyacrylamide gel electrophoresis using step 4 in 4.2.2;

[0069] 4. Use step 5 in 4.2.2 to read the GSA7.548 genotypes of various types according to the band height;

[0070] 5. Based on the 463, 455, and 1418 materials carrying the genotype AA and BB in the three generations of Yugu No. 1 × Setaria H1 (F2, F3, and F4), variance analysis was performed and it was found that GSA7.548 could classify the 463, 455, and 1418 millet progeny materials in F2, F3, and F4 into two types: those with few axillary branches and those with many axillary branches. Figure 3 , ANOVA bar chart.

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of a primer set in identifying the multi-leaf axillary branching trait of millet, characterized in that: The primer set includes a forward primer, whose nucleotide sequence is shown in SEQ ID NO.1; and a reverse primer, whose nucleotide sequence is shown in SEQ ID NO.2.

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