Indel marker related to antioxidant activity and total phenol content of sesame seed, and detection method and application thereof

Abstract

The application belongs to the technical field of sesame breeding, and discloses an Indel marker related to the antioxidant activity and total phenol content of sesame grains, a detection method and application thereof. The Indel marker is located at the position of 20822892 bp of the 6th chromosome of sesame, and the sequence characteristic is the deletion of a single base T (relative to the reference genome sequence of Zhishiba 13). Specifically, the primer InDelF / InDelR can be used to detect the Indel marker, and the obtained PCR amplification product is used Bsl I enzyme digestion and gel electrophoresis. The characteristic band of the marker linked to the high antioxidant activity and high total phenol content of sesame grains is a 428 bp band, and the characteristic band of the marker linked to the low antioxidant activity and low total phenol content of sesame grains is a 340 bp band. The Indel marker and the detection method provided by the application can be used for efficient screening of sesame germplasm resources with high grain antioxidant activity and total phenol content, and have important significance for the cultivation of new sesame varieties.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of sesame breeding, and particularly relates to an Indel marker related to the antioxidant activity and total phenol content of sesame seeds, a detection method thereof and application of the Indel marker in sesame germplasm screening. BACKGROUND

[0002] Sesamum indicum L. Sesamum indicum Sesamum indicum L. is an important and ancient oil crop, which is widely used in the fields of medicine, food, cosmetics and the like. Sesame is rich in unsaturated fatty acids, and contains various antioxidants such as sesamin, vitamin E, melatonin and phytosterols. Since ancient times, sesame has been considered as a good product for health preservation and health care. Modern medical research shows that the functional active substances in sesame seeds have various functions such as antioxidant, anti-diabetes, anti-hyperlipidemia, protection of kidney and liver, anti-inflammatory, protection of cardiovascular system, antihypertensive, anti-tumor and anti-cancer. The high antioxidant capacity of sesame enables it to be used for food antioxidant to improve the stability and quality of food.

[0003] The antioxidant capacity of sesame is affected by various factors such as production place, seed coat color and processing condition. Early research shows that black sesame has higher antioxidant capacity than other colored seeds. However, this conclusion is seriously challenged in a large population consisting of a large number of sesame with different colors. The lignans (sesamin, sesamolin and the like) unique to sesame are a kind of healthy antioxidant components. Some studies show that the change in seed antioxidant activity is only related to the difference in lignan content. However, some sesame with low lignan content has higher antioxidant activity than sesame with high lignan content. It is found through recent comparative analysis of the metabolome of sesame with different colors that the difference in relative content of certain phenolic compounds is the main reason for the difference in antioxidant activity.

[0004] Although there is evidence that the antioxidant capacity of sesame varies greatly, the potential biochemical and molecular basis thereof is still unclear. Identifying the antioxidant activity-related gene locus and developing a simple and easy molecular marker detection method have important significance for breeding new sesame varieties with high antioxidant activity, improving the nutritional quality of sesame and meeting the needs of people's healthy life. SUMMARY

[0005] Therefore, the application aims to develop a molecular marker related to the antioxidant activity trait of sesame seeds and an application method thereof, so as to accelerate the breeding process of new sesame varieties with high antioxidant activity.

[0006] The technical scheme of the application is as follows:

[0007] The first aspect of the present application provides an Indel marker related to the antioxidant activity and total phenol content of sesame seeds, taking Zhongzhi 13 as the reference genome, the Indel marker is located at 20822892 bp of the 6th chromosome of sesame (denoted as InDel6:20822892), which is manifested as the deletion of a single base T; and the allele analysis shows that the genotype consistent with the reference genome sequence is manifested as high antioxidant activity and high total phenol content of the seeds, and the genotype with the deletion of a single base T is manifested as low antioxidant activity and low total phenol content of the seeds.

[0008] In view of the lack of molecular markers related to the antioxidant activity of sesame seeds in the prior art, the inventors sampled 8690 domestic and foreign resources preserved by the National Sesame Medium-term Bank in stages, selected 160 sesame materials for resequencing analysis, and constructed a haplotype map of the association population according to the resequencing results, taking Zhongzhi 13 as the reference genome; then, combined with the detection results of the antioxidant activity and total phenol content of the sesame seeds, the whole genome association analysis was performed, and finally the InDel6:20822892 marker was mined. The marker is not only significantly associated with the antioxidant activity of the seeds, but also significantly associated with the total phenol content of the seeds, so that the identification and prediction of the two traits can be realized through the marker, which is beneficial to the screening of germplasm resources, and also indicates that the content of phenolic compounds in the seeds is one of the key factors affecting the antioxidant activity.

[0009] The second aspect of the present application provides primers for detecting the InDel6:20822892 marker, and in some embodiments of the present application, the detection primers are specifically:

[0010] InDelF: 5'-CCGCCCTTCAATGACACG-3';

[0011] InDelR: 5'-GATGGTTTTCCGCAGCAACT-3'.

[0012] The inventors found that the deletion of a single base T forms a recognition site of restriction enzyme Bsl I, while the corresponding reference genome sequence cannot be recognized by Bsl I by analyzing the flanking sequences of the InDel6:20822892 marker.

[0013] The third aspect of the present application provides a detection kit, which contains primers for detecting the InDel6:20822892 marker, and can also contain other reagents and consumables for PCR reaction.

[0014] The application provides the use of the InDel6:20822892 marker, primers or a kit for detecting the InDel6:20822892 marker, and is at least one of the following:

[0015] 1) used for identifying the antioxidant activity and / or total phenol content of sesame seeds;

[0016] 2) used for early prediction of the antioxidant activity and / or total phenol content of sesame seeds;

[0017] 3) used for sesame molecular marker-assisted breeding.

[0018] The application provides a method for detecting the antioxidant activity and total phenol content of sesame seeds, comprising the following steps:

[0019] S1, extracting genomic DNA of the sesame material to be detected;

[0020] S2, using the extracted genomic DNA as a template, performing PCR amplification on a DNA fragment containing the InDel6:20822892 marker;

[0021] S3, detecting the amplification product to obtain a genotype.

[0022] Preferably, in the above method, the primers InDelF / InDelR are used for PCR amplification in step S2, and step S3 is specifically: using the amplification product obtained in step S2 as a template, performing PCR amplification on the template using the primers InDelF / InDelR, and performing agarose gel electrophoresis on the amplification product to obtain a result. Bsl I digestion, and detecting the digestion product by agarose gel electrophoresis. Moreover, the result interpretation method is that the marker characteristic band linked to the high antioxidant activity and high total phenol content of sesame seeds is one and only one 428 bp band, and the marker characteristic band linked to the low antioxidant activity and low total phenol content of sesame seeds is one 340 bp band.

[0023] More preferably, in the above method, the PCR amplification system comprises a DNA template, 2×Taq PCR MasterMix solution, forward and reverse primers, and the rest of ddH2O.

[0024] More preferably, in the above method, the PCR amplification program is: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles; and 72℃ extension for 5 min.

[0025] Compared with the prior art, the application has the following beneficial effects:

[0026] The Indel marker significantly related to the antioxidant activity and total phenol content of sesame seeds is reported for the first time, and the detection method of the Indel marker and the application of the Indel marker in germplasm screening are further provided, which has important significance for rapidly screening excellent sesame germplasm with high antioxidant activity and high total phenol content, cultivating new sesame varieties with high antioxidant activity and high total phenol content, and improving the nutritional quality of sesame. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a Manhattan plot of whole genome association analysis of the antioxidant activity of sesame seeds in the embodiment 1 of the present application; wherein SD and HB are the association results of two different environmental phenotypes, and AOA represents antioxidant activity.

[0028] Figure 2 It is a Manhattan plot of whole genome association of the total phenol content of sesame seeds in the embodiment 1 of the present application; wherein SD and HB are the association results of two different environmental phenotypes, and TPC represents total phenol content.

[0029] Figure 3 It is a sequence alignment plot of two genotypes of the InDel6:20822892 marker; Ref represents the InDel6:20822892 and the reference genome sequence of Zhizhi 13 consistent genotype, and Alt represents the InDel6:20822892 single base T deletion genotype.

[0030] Figure 4 It is a gel electrophoresis plot of the InDel marker PCR amplification product of different sesame materials in the embodiment 3 of the present application after I digestion; M is marker, 1-5 are five high antioxidant activity and high total phenol content materials, and 6-10 are five low antioxidant activity and low total phenol content materials. Bsl I digestion; M is marker, 1-5 are five high antioxidant activity and high total phenol content materials, and 6-10 are five low antioxidant activity and low total phenol content materials.

[0031] Figure 5 It is the frequency distribution of antioxidant activity and total phenol content in 200 sesame germplasm resources in the embodiment 4 of the present application; AOA represents antioxidant activity, and TPC represents total phenol content.

[0032] Figure 6 It is the comparison of antioxidant activity and total phenol content of different genotype materials of the InDel6:20822892 marker in 200 sesame germplasm resources in the embodiment 4 of the present application; AOA represents antioxidant activity, TPC represents total phenol content, Ref represents the InDel6:20822892 and the reference genome sequence of Zhizhi 13 consistent genotype, and Alt represents the InDel6:20822892 single base T deletion genotype. DETAILED DESCRIPTION

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The term "comprising" and variations thereof as used in the present specification and claims are intended to cover the inclusive meaning of the term, but not the exclusive meaning.

[0034] The technical solutions of the present application will be described clearly and completely in combination with specific embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0035] In the following examples, the specific techniques or conditions not specified are all according to the techniques or conditions described in the literature in the art or according to the product instructions; the reagents or instruments not specified by the manufacturer are all conventional products that can be obtained by purchase.

[0036] Example 1

[0037] This example provides an Indel marker significantly associated with the antioxidant activity and total phenol content of sesame seeds, which is located at the position of 20822892 bp of the 6th chromosome of sesame and the sequence feature is the deletion of a single base T, with "Zhongzhi 13" as the reference genome. The way to obtain the Indel marker of the present application includes the following steps:

[0038] (1) Construction of association population, resequencing and haplotype map construction.

[0039] From the 8690 domestic and foreign resources preserved by the National Sesame Medium-term Library, according to the results of geographical origin, phenotype and genetic diversity detection, 160 sesame materials were selected for resequencing analysis by using a step-by-step sampling strategy. Using the Illumina Hiseq2500 sequencing platform, the 160 sesame materials were subjected to high-coverage whole genome resequencing by using double-end sequencing method, and the genome sequences with an average coverage of more than 20 times were obtained. Taking "Zhongzhi 13" as the reference genome, the haplotype map of the association population was constructed, containing 1.39 million SNP markers and 0.25 million InDel markers.

[0040] (2) Detection of antioxidant activity and total phenol content of seed kernels of association population materials.

[0041] The above 160 sesame materials were planted in Linqing, Shandong and Wuhan, Hubei, respectively, and the antioxidant activity and total phenol content of the harvested kernels were detected. The antioxidant activity was represented by the DPPH (1,1-diphenyl-2-trinitrobenzene hydrazine) free radical scavenging rate, and the total phenol content was determined by the Folin phenol method, and the specific methods were as follows:

[0042] Preparation of sesame seed extract: Weigh 0.5 g of seeds, add 5 mL of 80% ethanol, homogenize, and extract by shaking for 6 h. Centrifuge at 10,000 r / min for 10 min, and the supernatant is the sesame seed extract.

[0043] DPPH free radical scavenging rate determination: 10 μL of sesame seed extract was mixed with 190 μL of 0.2 mM DPPH ethanol solution in a 96-well plate and incubated in the dark at room temperature for 30 min; the absorbance was measured at 515 nm using a microplate reader. The control group used anhydrous ethanol instead of DPPH, and the blank group used anhydrous ethanol instead of the sample. The DPPH free radical scavenging rate (%) was calculated as: [1-(A 样品 -A 对照 ) / A 空白 ]×100.

[0044] Total phenol content determination: Add 400 μL dH2O and 100 μL Folin-Ciocalteu reagent to 100 μL sesame seed extract, mix thoroughly; let stand for 6 min, then add 1 mL 7% (m / v) Na2CO3; after standing at room temperature for 90 min, measure the absorbance at 760 nm. Use 80% ethanol as a blank control instead of the sample. Total phenol content is expressed as gallic acid equivalents (mg gallic acid equivalents / g), and the standard curve equation is y = 1.971x-0.0068, R 2 = 0.99.

[0045] (3) Genome-wide association analysis of grain antioxidant activity and total phenol content.

[0046] Genome-wide association analysis (GWA) of antioxidant activity and total phenolic content in sesame seeds from two environments in Hubei and Shandong provinces was performed using the rMVP software package with a mixed linear model. In both environments, an InDel located at 20822892 bp on chromosome 6 (denoted as InDel6:20822892) was detected and significantly associated with seed antioxidant activity (Shandong environment: P = 2.64×10 -11 Hubei Environment: P = 1.46×10 -8 )(like Figure 1 As shown in the figure, it explained 33.31% and 19.87% of the phenotypic variation, respectively. It was also found that InDel6:20822892 was significantly associated with the total phenolic content of grains under both environments (Shandong environment: P = 2.69×10 -10 Hubei Environment: P = 3.69×10 -7 )(like Figure 2The InDel6:20822892 marker was developed in this example. The InDel6:20822892 marker explained 26.50% and 12.32% of the phenotypic variation of the seed total phenol content and the seed antioxidant activity, respectively.

[0047] The InDel6:20822892 marker showed a deletion of a single base T compared with the reference genome of Zhizhi 13. Figure 3 The genotype with the sequence consistent with the reference genome showed high seed antioxidant activity and high total phenol content, while the genotype with the deletion of the single base T showed low seed antioxidant activity and low total phenol content.

[0048] Example 2

[0049] For the InDel6:20822892 marker provided in the present application, the primer and method for detecting the Indel marker were developed in this example.

[0050] (1) Primer for detecting the Indel marker.

[0051] The sequence analysis of the flanking sequence of the InDel6:20822892 marker showed that the deletion of the single base T formed a recognition site (5'-CCNNNNNNNGG-3') for the restriction enzyme I, while the corresponding reference genome sequence could not be recognized by the restriction enzyme I. Therefore, the InDel marker was developed as a CAPS marker in this example to facilitate the genotype detection of the InDel marker. Specifically, the primer sequences provided in this example are as follows: Bsl Bsl InDelF: 5'-CCGCCCTTCAATGACACG-3' (SEQ ID NO. 1);

[0052] InDelR: 5'-GATGGTTTTCCGCAGCAACT-3' (SEQ ID NO. 2).

[0053] (2) Method for detecting the Indel marker.

[0054] Based on the primer provided in step (1), the method for detecting the Indel marker comprises the following steps:

[0055] ① Extraction of genomic DNA;

[0056] ② Using the extracted genomic DNA as a template, the DNA fragment containing InDel6:20822892 is amplified by PCR using the primers InDelF / InDelR;

[0057] ③ The PCR product generated by amplification is digested with the restriction enzyme I;

[0058] ④ The digestion results are analyzed to determine the genotype of the InDel6:20822892 marker. Bsl ​I enzyme digestion, and then agarose gel electrophoresis was used to detect the enzyme digestion products; wherein the material with high antioxidant activity and high total phenol content should have a 428 bp band, and the material with low antioxidant activity and low total phenol content should have a 340 bp band.

[0059] Example 3

[0060] In this example, 5 high antioxidant activity and high total phenol content materials (InDel6:20822892 is consistent with the reference genome sequence) and 5 low antioxidant activity and low total phenol content materials (InDel6:20822892 is a single base T deletion) were selected from the 160 sesame materials described in Example 1 to verify the feasibility of the method of Example 2. Specifically, the following operations were included:

[0061] (1) Extraction of genomic DNA.

[0062] The CTAB plant genomic DNA rapid extraction kit (Wuhan Junnuode Biotechnology Co., Ltd.) was used to extract the genomic DNA of the sesame sample, and the kit instructions were followed.

[0063] (2) Amplification of the DNA fragment containing InDel6:20822892.

[0064] The genomic DNA extracted in step (1) was used as a template, and the DNA fragment containing InDel6:20822892 was amplified by PCR using primers InDelF / InDelR. The PCR amplification system was 10 μL, including DNA template 0.5 μL, PC09-2×Taq PCR MasterMix (Beijing Aidley Biotechnology Co., Ltd.) 4 μL, forward and reverse primers each 0.2 μL, and ddH2O was used to make up the total volume to 10 μL. The PCR amplification program was: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles; 72℃ extension for 5 min.

[0065] (3) Enzymatic digestion and gel electrophoresis of the PCR product.

[0066] The PCR product produced in step (2) was digested with Bsl I, and the enzyme digestion system was: restriction endonuclease Bsl I 0.5 μL, 10×Buffer Tango buffer 1 μL, PCR product 5 μL, and ddH2O was used to make up the total volume to 10 μL. 55℃ enzyme digestion for 4h, 65℃ inactivation for 20 min.

[0067] The enzyme digestion products were detected by 1.2% agarose gel electrophoresis, and the detection results were as follows Figure 4As shown, the material with high antioxidant activity and high total phenol content has only the 428 bp band, and the material with low antioxidant activity and low total phenol content has the 340 bp band after enzyme digestion.

[0068] Example 4

[0069] This example verifies the application of the InDel6:20822892 marker in screening of sesame germplasm resources with high antioxidant activity and high total phenol content, and the experimental steps are as follows:

[0070] Randomly select 200 materials from the germplasm resource library, and detect the antioxidant activity and total phenol content of the grains by the method described in step (2) of Example 1. The results show that the antioxidant activity and total phenol content of the 200 materials are normally distributed (P > 0.05). Figure 5 ).

[0071] Genotype the InDel6:20822892 marker by the detection method in Example 3. The results show that among the 200 materials, the genotype of the InDel6:20822892 marker consistent with the reference genome sequence is 164, and the genotype of single base T deletion is 36; the comparison of phenotypes of different genotypes shows that the antioxidant activity and total phenol content of the genotype consistent with the reference genome are significantly higher than those of the genotype of single base T deletion (P < 0.001). P <0.001). Figure 6 ).

[0072] In summary, the InDel marker InDel6:20822892 provided by the present application is significantly associated with the sesame grain antioxidant activity and total phenol content traits, and the detection method developed by the present application has the characteristics of low cost, less limitation, high accuracy and high efficiency when screening sesame germplasm resources with high antioxidant activity and high total phenol content.

[0073] It should be noted that the above examples are only a part of the embodiments of the present application but not all the embodiments, and are used to illustrate the technical solutions of the present application but not to limit; based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

Claims

1. Use of an Indel marker primer or a kit comprising said primer, characterized in that, Any one of the following: 1) for identifying the traits of antioxidant activity and / or total phenol content of sesame seeds; 2) for early prediction of the traits of antioxidant activity and / or total phenol content of sesame seeds; With "Zhizhi 13" as the reference genome, the Indel marker is located at 20822892 bp of the 6th chromosome of sesame, showing the deletion of a single base T; the genotype consistent with the reference genome sequence shows high antioxidant activity and high total phenol content of the seeds, and the genotype with the deletion of a single base T shows low antioxidant activity and low total phenol content of the seeds, the primers of the Indel marker include: InDelF: 5'-CCGCCCTTCAATGACACG-3' and InDelR: 5'-GATGGTTTTCCGCAGCAACT-3'.

2. A method for identifying the traits of antioxidant activity and total phenolic content of sesame seeds, characterized by, The steps include: S1, extracting the genomic DNA of the sesame material to be tested; S2, using the extracted genomic DNA as a template to perform PCR amplification on the DNA fragment containing the Indel marker of claim 1; S3, detecting the amplification product to obtain the genotype.

3. The method of authentication according to claim 2, wherein, In step S2, the primers InDelF / InDelR are used for PCR amplification, the InDelF is 5'-CCGCCCTTCAATGACACG-3', and the InDelR is 5'-GATGGTTTTCCGCAGCAACT-3'.

4. The method of authentication according to claim 3, characterized in that, Step S3 specifically involves: processing the amplification product obtained in step S2 using... Bsl Enzyme I digestion was performed, and the digestion products were detected by agarose gel electrophoresis. Among them, the marker characteristic band linked to the traits of high antioxidant activity and high total phenol content of sesame seeds was a single 428 bp band, and the marker characteristic band linked to the traits of low antioxidant activity and low total phenol content of sesame seeds was a single 340 bp band.

5. The method of authentication according to claim 3, wherein, The system of the PCR amplification includes a DNA template, 2×Taq PCR MasterMix solution, forward and reverse primers, and the rest of ddH2O.

6. The method of authentication according to claim 3, wherein The program of the PCR amplification is: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles; 72℃ extension for 5 min.

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