Application of Yellow Seed 2 Gene and Its Indel-Marked Primer Pairs in Controlling Yellow Seed Traits in Sorghum

Abstract

The present invention belongs to the technical field of molecular marker-assisted breeding, specifically discloses the application of Yellow Seed2 gene and primer pairs marked with Indel in controlling yellow grain traits of sorghum, discloses the Yellowseed2 gene sequence for controlling grain color traits of sorghum, first observes grain color changes, and further performs genetic positioning, candidate gene screening and expression analysis. Sequencing shows that three CTG bases are inserted at the 619bp-621bp position in the coding region of Yellow Seed2 (Sobic.001G397900) in the two materials 699 and GW001938, resulting in an additional leucine, so it is speculated that Yellow Seed2 may be involved in the formation of grain colors of these two parent materials; Indel markers and primer pairs are developed using Yellow Seed2 sequences, and bands of different fragment sizes appear using polyacrylamide electrophoresis, so that sorghum grain color can be quickly identified.

Description

Technical Field

[0001] The invention relates to the technical field of molecular marker-assisted breeding, in particular to the application of Yellow Seed 2 gene and primer pairs of Indel markers thereof in controlling yellow grain traits of sorghum. Background Art

[0002] Sorghum plays an important role in my country's agricultural structural adjustment and brewing industry. Grain color is one of the important traits, but there are not many studies on sorghum grain color.

[0003] There are reports abroad that Yellow Seed1 controls sorghum grain color, but it is not clear in China whether there are other genes that control sorghum grain color. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the use of YellowSeed2 gene and its Indel-marked primer pair in controlling the yellow grain trait of sorghum.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] One of the purposes of the present invention is to provide an application of Yellow Seed2 gene in controlling yellow grain trait of sorghum, characterized in that the nucleotide sequence of the Yellow Seed2 gene is shown as SEQ ID NO.1.

[0007] The second object of the present invention is to provide an Indel-marked primer pair for detecting a gene for the yellow seed trait of sorghum, the primer pair comprising an upstream primer as shown in SEQ ID NO.2 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.3; wherein the polymorphic site of the Indel marker is located at 619bp-621bp in the Yellow Seed2 gene coding region, and three bases CTG are inserted between 619bp-621bp.

[0008] A fourth object of the present invention is to provide an application of an Indel-labeled primer pair for detecting a gene for the yellow grain trait of sorghum in identifying the color of sorghum grains.

[0009] Compared with the prior art, the present invention discloses a Yellow seed2 gene sequence for controlling sorghum grain color traits. First, the grain color change is observed, and then genetic positioning, candidate gene screening and expression analysis are performed. Sequencing shows that three bases CTG are inserted at the 619bp-621bp position in the coding region of Yellow Seed2 (Sobic.001G397900) in two materials, 699 and GW001938, resulting in an additional leucine. Therefore, it is speculated that Yellow Seed 2 may be involved in the formation of the grain color of the two parent materials. Indel markers and primer pairs are developed using the Yellow Seed2 sequence, and bands of different fragment sizes appear using polyacrylamide electrophoresis, so that the sorghum grain color can be quickly identified. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the process of yellow grain development of sorghum.

[0011] Figure 2 is the distribution of SNP-index association values ​​on chromosomes.

[0012] Figure 3 To finely map the gene controlling the yellow grain trait in sorghum.

[0013] Figure 4 : a is the agarose electrophoresis diagram of YellowSeed 1 in three materials, 699, GW001938, and BTX623; b is the sequencing peak diagram of YellowSeed2 in two materials, 699 and GW001938.

[0014] Figure 5 Expression analysis of candidate genes.

[0015] Figure 6 Develop applications for Indel markers. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0017] 1. Source of test materials

[0018] Parental material 699 (yellow grain material) and GW001938 (white grain material) are natural population materials preserved by our research group through self-pollination for many years (the labels of these two materials are derived from the "National Sorghum Variety Resource Catalog" and the "China Sorghum Variety Resource Catalog"). F1 bagged self-pollination, 3215 individual plants of F2 genetic segregation population were obtained. Planted at the experimental base of Liaoning Academy of Agricultural Sciences, the two parents were planted in 3 rows respectively, and the F2 segregation population was planted in about 95 rows, with a row length of 8m, a plant spacing of 0.25m, and a row spacing of 0.6m, with conventional field management.

[0019] 2. Identification of morphological changes in yellow sorghum grain development

[0020] During the flowering period, 699 plants were marked with a combination of hanging tags, gauze bags and markers, with 5 plants marked every day. Sorghum grains of different development days were collected and placed under a stereo microscope. The morphology of fresh grains was obtained by precise focusing with a photographic eyepiece and objective lens. During the entire development process of sorghum grains, 1-16 days is a rapid growth period, 17-24 days enters a slow expansion period, and the 25th day begins to enter a color change period, the green color gradually fades, and the grain color gradually changes from milky white or pale to light yellow, and then the color deepens. After 40 days, the grain color becomes dark yellow (see Figure 1 ).

[0021] 3. Improved CTAB DNA extraction

[0022] (1) Select 50 fresh leaves from plants with yellow seed phenotype and 50 fresh leaves from plants with white seed phenotype, place them in 2.0 ml centrifuge tubes, grind them into powder using a tissue grinder at low temperature, and store them at -80°C for later use.

[0023] (2) Before melting the sample, add 1 mL of CTAB preheated at 65°C, mix thoroughly by inversion, and place in a metal bath at 65°C for 30 min, mixing thoroughly by inversion every 10 min.

[0024] (3) Add 0.7 ml of chloroform at room temperature, mix vigorously, centrifuge at 12,000 rpm for 5 min, transfer the supernatant to a sterilized 1.5 ml centrifuge tube, add 0.5 ml of chloroform, and mix.

[0025] (4) Centrifuge at 12000 rpm for 10 min, take the supernatant and transfer it to a 1.5 ml centrifuge tube. Add an equal volume of -20°C pre-cooled isopropanol and gently mix. At this time, a white flocculent precipitate visible to the naked eye will appear in the centrifuge tube.

[0026] (5) Incubate at -20°C for 20 min, centrifuge at 12,000 rpm for 10 min, and discard the supernatant.

[0027] (6) Add 1 ml of pre-cooled 75% alcohol, shake gently to float the precipitate, and centrifuge at 12,000 rpm for 3 min.

[0028] (7) Repeat the previous step and discard the supernatant.

[0029] (8) Briefly centrifuge and remove the residual alcohol with a pipette. Place the tube on a clean bench to air-dry until the DNA is completely transparent. Add 150 ml of RNase A-free sterile water. After the DNA is fully dissolved, incubate at 37°C for 30 min.

[0030] (9) Agarose gel electrophoresis was used to detect whether there was RNA contamination. If there was no RNA contamination, the concentration and quality of the DNA were tested using a UV spectrophotometer. When the 260 / 280 ratio was between 1.8 and 2.0, it indicated that the DNA was not contaminated. If the 260 / 280 ratio was lower than 1.8, it indicated that there was protein contamination; if the 260 / 280 ratio was higher than 2.0, it indicated that there was RNA contamination. The qualified DNA samples were diluted to 100 ng / ul and stored at -20°C for later use.

[0031] 4. Initial and fine localization of YellowSeed2 gene

[0032] 4.1 Preliminary localization of the gene Yellow Seed2 controlling the yellow grain trait in sorghum

[0033] From the F2 segregating population of the natural population 699×GW001938, 50 fresh leaves of plants with yellow grain phenotype and 50 fresh leaves of plants with white grain phenotype were selected, and genomic DNA was extracted by the modified CTAB method. The qualified genomic DNA was mixed in equal amounts to construct mixed pools. The genomic DNA was mechanically broken, purified, end-repaired, A-added, and adapter-added, and then fragment selection and PCR enrichment were performed after agarose electrophoresis. A sequencing library was constructed, and the library quality was checked after construction. The qualified library was resequenced using the Illumina platform to preliminarily locate the gene Yellow Seed2 that controls the yellow grain trait. According to the resequencing results of sorghum in the F2 segregating population constructed by 688 and GW001938, the gene Yellow Seed2 that controls the yellow grain trait of sorghum was located within 15.4Mb on chromosome 1 (see Figure 2 ).

[0034] 4.2 Fine mapping of the Yellow Seed2 gene controlling the yellow grain trait in sorghum

[0035] According to the preliminary positioning results, BTX623 was used as the reference genome to screen markers for polymorphism between parents, and the selected polymorphic markers were used to finely locate 3215 individuals in the F2 population. Combined with the information of multiple sorghum genome sequencing varieties published on the sorghum gene annotation website, 8359 pairs of SSR and Indel molecular markers were developed for the preliminary positioning interval. The selected polymorphic markers AR5, AR6, BR2, BR13, BR18, BR20, AR8, AR9 and AR16 were used to finely locate 3215 individuals in the F2 segregation population, further narrowing the positioning interval. The results showed that there were 200 exchanged individuals between the BR13 and BR18 markers, and Yellow Seed 2 was initially located between these two markers, with a physical distance of 0.54Mb. To further narrow the positioning interval, the present invention developed SSR and Indel markers between BR13 and BR18: P2, P5, P7 and P8, and finally located the gene Yellow Seed2 controlling the yellow seed trait within the 108 kb range between BR13 and P2 markers (see Figure 3 and Table 1).

[0036] Table 1 Primer sequences

[0037]

[0038] 4.3 PCR amplification

[0039] The total volume of the amplification reaction was 10 μL: 1 ul DNA, 5 ul 2×PCR premix, and 5 ul ddH2O. The PCR amplification reaction program was: 95°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 35 cycles, and 72°C extension for 10 min. The PCR product was subjected to 8% polyacrylamide gel electrophoresis, and photographed after development.

[0040] 4.4 Polyacrylamide electrophoresis

[0041] (1) Gel preparation

[0042] Wash the glass plate repeatedly with ultrapure water, wipe it with 70% ethanol, dry it, and assemble the glass plate. Add 20mL of 30% gel stock solution, 8mL of 5xTBE solution, 880uL of 10% APS solution, 66uL of TEMED, 9g of urea, and 52mL of ddH2O in a beaker, mix well, and quickly inject into the gap of the glass plate to prevent bubbles. After the gel is poured, insert the gel comb, wipe the overflowed gel, and polymerize for about 1 hour. After the gel is solidified, pull out the comb and assemble the electrophoresis tank.

[0043] (2) Gel electrophoresis

[0044] Inject 0.5xTBE electrophoresis buffer into the electrophoresis tank, put the film into the electrophoresis tank, avoid bubbles in the bottom gap when putting the film, inject 0.5xTBE electrophoresis buffer into the top of the electrophoresis tank to exceed 1.5cm above the top of the film, and remove the comb. Add 1uL PCR product to each well, turn on the power supply, voltage 135V, current 400mA, and the electrophoresis time is determined by the size of the amplified fragment.

[0045] (3) Silver staining

[0046] After the electrophoresis is finished, place the glass plate in distilled water, carefully separate the two glass plates, and place the film close to the long glass plate. Carefully remove the film from the distilled water and immediately place it in the pre-configured 0.5% silver nitrate solution staining solution. Shake it gently on a shaker for 10 minutes. Take out the film from the staining solution and wash it once with distilled water for no more than 10 seconds. Wash away the residual AgNO3 solution on the film surface with AgNO3. Avoid excessive washing to prevent unclear or no bands during color development.

[0047] (4) Development and photography

[0048] Place the washed film in the color developing solution and shake it gently until the strips are clear. After the color development is completed, use distilled water to wash away the residual color developing solution, lay the film flat under a fluorescent light box, write the number, and take a photo with a camera for storage.

[0049] 5. Candidate gene prediction

[0050] In order to identify the gene controlling yellow grain, we searched for all genes and their functional annotations within the 104 kb interval on the sorghum gene annotation website (http: / / plants.ensembl.org / Sorghum_bicolor / Info / Index) and found that there were 7 candidate genes in this interval, including 6 known protein-coding genes and 1 gene of unknown function (see Table 2).

[0051] Table 2 Candidate gene name, location, function

[0052]

[0053]

[0054] The two parents, 699 and GW001938, were sequenced and verified. The nucleotide sequences of Sobic.001G397600, Sobic.001G397850, and Sobic.001G398000 did not change. There were 5 SNP differences in the nucleotide sequences of Sobic.001G397700 and Sobic.001G397800, but the amino acid sequences did not change. Yellow Seed 1 is related to MYB domain protein 11, a gene that regulates the color of sorghum kernel pericarp. Pan-genome analysis revealed that the 5' untranslated region and the first two exons of this gene were missing in four sorghum varieties, including the sequenced variety BTX623, leading to the loss of function of the gene. Agarose electrophoresis confirmed that the Yellow Seed 1 gene in the two parental materials in this study lacked the 5' untranslated region and the first and second exons, just like the BTX623 material (see Figure 4 ), since the grains of the two parental materials are white and yellow respectively, it is believed that the difference in grain color is not regulated by Yellow Seed 1, but by another MYB-like transcription factor Yellow Seed2 in the candidate interval. Sequencing results show that three bases CTG are inserted at 619bp-621bp in the Yellow Seed2 coding region, resulting in an additional amino acid L (see Figure 5 ). Therefore, YellowSeed2 is considered to be a new gene regulating sorghum grain color.

[0055] 6. Expression analysis of Yellowseed2 gene controlling yellow grain trait in sorghum

[0056] The expression levels of 7 candidate genes in the candidate interval were measured by real-time quantitative experiments (see Table 2). After analyzing and processing the data, it was found that the Yellowseed2 gene had the highest expression level in mature seeds, and the expression level of Yellowseed2 in 688 was significantly higher than that in GW001938. This may be because Yellowseed2 positively regulates the production of flavonoids and thus affects the change in sorghum grain color. Therefore, the difference in grain color between the two materials is regulated by Yellowseed2 among the 7 candidate genes.

[0057] 7. Indel marker development

[0058] 7.1. Indel marker design

[0059] Indel markers were designed using the NCBI website (BLAST: Basic Local Alignment Search Tool (nih.gov)) combined with the Yellowseed2 genome sequence.

[0060] 7.2 PCR reaction

[0061] (1) Using more than 600 natural population materials, genomic DNA was extracted. Specific implementation method (3. Improved CTAB method DNA extraction).

[0062] (2) PCR reaction system (10 μl):

[0063] DNA 1μl, upstream primer SEQ ID NO.2 0.5μl, downstream primer SEQ ID NO.3 0.5μl, 2×PCR premix 5μl, H2O 5ul.

[0064] (3) PCR reaction procedure:

[0065] Denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 35 cycles, extension at 72°C for 10 min, and storage at -20°C.

[0066] 7.3. Indel molecular marker verification

[0067] The PCR products were subjected to polyacrylamide electrophoresis, silver staining, development and photography to analyze the Indel markers and verify the validity of the Indel molecular markers (see Figure 6 ).

Claims

1. A primer pair for detecting an Indel marker gene for yellow grain trait of sorghum, characterized in that: The primer pair includes an upstream primer as shown in SEQ ID NO.2 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.3; wherein the polymorphic site of the Indel marker is located at Yellow Seed 2 At 619bp-621bp in the gene coding region, three bases CTG are inserted between 619bp-621bp; Yellow Seed 2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Use of the Indel-tagged primer pair for detecting a gene for yellow grain trait of sorghum as claimed in claim 1 in identifying sorghum grain color.

Images