InDel molecular marker of avena sativa and application of InDel molecular marker

Abstract

The invention relates to the technical field of crop molecular genetic markers, and discloses an InDel molecular marker of avena nuda and application thereof.The molecular marker is a group of 314bp deletion variation and 2bp insertion variation in the 610439484-610440026 interval of the 5C chromosome position of a reference genome of an avena nuda variety OT3098, the nucleotide sequence of the deletion variation is shown as SEQ ID NO.1, the nucleotide sequence of the insertion variation is shown as SEQ ID NO.2, and the nucleotide sequence of the insertion variation is shown as SEQ ID NO.3. The nucleotide sequence of the insertion variation is 5 '-AA-3'. The molecular marker provided by the invention can realize stable detection in agarose gel electrophoresis by combining a specific primer pair (SEQ ID NO.2-3) with an optimized touchdown PCR (Polymerase Chain Reaction) program, has the advantage of being not influenced by a tissue part and a growth period, and provides an efficient and reliable technical means for oat germplasm identification, seed purity detection and molecular breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of crop molecular genetic markers, and in particular to an InDel molecular marker of oats and its application. Background Art

[0002] Molecular markers can directly reflect genetic differences through DNA sequence polymorphism and have significant advantages over traditional morphological markers: (1) they are not affected by developmental stages and the environment; (2) they have high detection throughput and good repeatability; and (3) they can cover whole-genome variations. InDel markers developed based on next-generation sequencing technology have the characteristics of high polymorphism and strong stability: their insertion / deletion variations are densely distributed in the genome and are stable under different genetic backgrounds. They have become a core tool for crop genetic research and play a key role in the positioning of important traits and molecular breeding. In recent years, the development of next-generation sequencing technology has obtained a large amount of genetic data of plant variety resources in extensive resequencing or simplified genome sequencing, and has promoted the mining of whole-genome insertion / deletion length polymorphism markers and the precise analysis of genetic differences between varieties. InDel molecular markers belong to the third generation of molecular markers. Compared with traditional molecular markers such as RFLP, RAPD, AFLP, and SSR, they have high polymorphism: they cover the whole genome, are densely distributed, and cover coding and non-coding regions; there are significant differences between different individuals. Strong stability: they are not prone to mutation, and they are highly reliable and repeatable in multiple generations and different environments, and the results are intuitive. Therefore, InDel molecular markers have broad application potential in molecular marker-assisted breeding and precise positioning of important traits.

[0003] The naked husk of oats is a key agronomic trait, significantly impacting seed quality, yield, and processing methods. The primary difference between husk and naked oats lies in the ease with which the husk can be separated from the kernel after maturity. Husk oats retain their husk upon maturity, with a leathery, hard lemma attached to the caryopsis. Naked oats, on the other hand, retain their husk upon maturity, with a membranous, soft lemma that easily separates from the caryopsis. Currently, the identification of husk and naked oats relies primarily on traditional morphological analysis, which relies on observing the appearance of the kernel (whether the kernel has a husk, spikelet structure, etc.). However, the morphological differences between husk and naked oats are not obvious during the seedling and vegetative stages, requiring accurate differentiation at maturity. This can take several months, making conventional morphological methods difficult to achieve rapid and early identification, severely limiting breeding efficiency and seed quality testing. Furthermore, relying solely on morphological observation is susceptible to observer experience and subjective factors, leading to misjudgment, especially when the kernel's appearance is not obvious. Furthermore, morphological observation requires kernel-by-kernel inspection, which is time-consuming and labor-intensive, making it inefficient, particularly in large-scale breeding and variety identification. Therefore, developing an efficient, accurate, and easy-to-use method for identifying hulled / naked oats is of great significance. Summary of the Invention

[0004] In light of this, the present invention aims to provide an InDel molecular marker for hulled oats and its application. This technology aims to utilize InDel molecular marker technology to rapidly and accurately identify hulled and naked oats, establishing a rapid and accurate genotyping system. This technology has significant theoretical value and application prospects for improving the efficiency of germplasm resource management, ensuring seed quality and safety, and accelerating the genetic improvement of oats. This invention can promote the transition and upgrading of oat breeding from traditional empirical models to modern molecular design breeding.

[0005] The purpose of the present invention is achieved through the following technical solutions: In a first aspect, the present invention provides an InDel molecular marker of oat, which is a group of 314 bp deletion variations and 2 bp insertion variations located in the 610439484-610440026 interval of chromosome 5C of the reference genome Oat_OT3098_v2 (NCBI Assembly GCA_022788535.1) of the oat variety OT3098. The deletion sequence is shown in SEQ ID NO.1, and the insertion sequence is 5'-AA-3'.

[0006] Based on whole-genome resequencing technology, the present invention successfully screened out an InDel molecular marker site interval that can significantly distinguish between hulled and naked oats based on sequence alignment and variant site analysis of representative samples of hulled and naked oats. The site interval is located in the 610439484-610440026 interval of chromosome 5C of the hulled oat variety OT3098 reference genome Oat_OT3098_v2 (NCBI AssemblyGCA_022788535.1). Based on this site interval, an InDel molecular marker was designed and named OA-InDel-133. The molecular marker OA-InDel-133 includes a 314 bp deletion variation (nucleotide sequence shown in SEQ ID NO.1) and a 2 bp insertion variation (nucleotide sequence of 5'-AA-3"). A stable 312 bp length fragment difference exists between hulled and naked oats.

[0007] Furthermore, the present invention designed a pair of specific primers based on the InDel molecular marker site interval (forward primer F: 5'-CAACTTAACTTCTATGTTGAC-3' (SEQ ID NO. 2), reverse primer R: 5'-CAAACTAGCTTTTCTAACAA-3' (SEQ ID NO. 3)). By optimizing the PCR reaction system and touchdown amplification procedure, a 231 bp specific band can be stably amplified in hulled oats, while naked oats amplified a 543 bp main band or no amplification product (no 231 bp specific main band) under these primers.

[0008] In a second aspect, the present invention provides a method for detecting the InDel molecular marker, wherein the detection method is to perform PCR amplification using the specific primer pair designed by the present invention, and the molecular marker amplification product is identified by electrophoresis as a 231 bp band on an agarose gel.

[0009] Furthermore, the nucleotide sequence of the 231 bp band is shown in SEQ ID NO. 4, wherein Y / S / R / M / K are non-conserved base sites.

[0010] Furthermore, the PCR amplification system comprises 0.2-1.0 µL of DNA template, 10 µL of 2×TaqMaster Mix, 1 µL of the forward primer according to claim 2, 1 µL of the reverse primer according to claim 2, and the balance is ddH2O per 20 µL.

[0011] Furthermore, the PCR amplification program is a touchdown PCR program, and the program reaction conditions are: pre-denaturation at 94-95°C for 3-5 minutes; followed by two-stage cycling: the first stage is denaturation at 94-95°C for 15-40 seconds, annealing at 55-62°C for 15-40 seconds, and extension at 72°C for 15-40 seconds, for a total of 6-12 cycles; the second stage is denaturation at 94-95°C for 15-40 seconds, annealing at 50-55°C for 15-40 seconds, and extension at 72°C for 15-40 seconds, for a total of 25-35 cycles; finally, extension at 72°C for 5 minutes and storage at 4°C.

[0012] In a third aspect, the present invention seeks to protect the application of the InDel molecular marker or its detection method in the identification of oat germplasm resources.

[0013] Furthermore, the oat germplasm identification method involves extracting genomic DNA from the test sample as a template, using the specific primer pairs provided by the present invention for PCR amplification and electrophoresis identification. Electrophoresis results indicate that a 200-300 bp band in the electrophoresis lane indicates oats, while a 500-600 bp band or no characteristic band indicates naked oats. Experimental validation demonstrated that the InDel molecular marker demonstrated good specificity and stability across 48 test samples (see Table 1, including 23 naked oat varieties and 25 oat varieties), exhibiting excellent interspecies discrimination and high reproducibility, with an identification accuracy rate of 100%.

[0014] Compared with the prior art, the present invention has the following beneficial technical effects: (1) The InDel molecular marker OA-InDel-133 provided by the present invention has high specificity and stability, and can accurately distinguish between hulled oats and naked oats based on insertion and deletion differences at the genomic level. It overcomes the problems of traditional morphological methods that are highly dependent on growth period and environmental conditions and have poor accuracy, and significantly improves the efficiency and accuracy of germplasm resource identification; (2) The InDel molecular marker OA-InDel-133 provided by the present invention has high specificity and good repeatability, and the amplification results are stable and reliable. It is not affected by the sample tissue type, growth period and environmental conditions. It is suitable for a variety of application scenarios such as germplasm resource classification, variety identification, seed purity detection, and breeding material traceability. It has important promotion and application value and industrialization prospects. (3) The primer pairs and their supporting PCR system and touchdown amplification procedure provided by the present invention have good amplification stability and applicability, are not restricted by sample tissue type and cultivation conditions, and can repeatedly obtain consistent amplification results under different experimental conditions. They are suitable for various application scenarios such as seed purity detection, germplasm resource management, hybrid breeding screening, and protection of new plant variety rights, and have broad practical promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0016] Figure 1 This is the electrophoresis diagram of PCR amplification of oat materials No. 1-24 in Example 2 of the present invention; Figure 2 This is the electrophoresis diagram of PCR amplification of oat materials No. 25-48 in Example 2 of the present invention. DETAILED DESCRIPTION

[0017] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0018] Unless otherwise specified, the reagents and equipment used in the following examples can be purchased through regular commercial channels.

[0019] Example 1. Design of InDel molecular markers and primers Based on the reference genomes ASM2364667v1 (NCBI Assembly GCA_023646675.1) of the hexaploid cultivated naked oat variety "Sanfensan" and Oat_OT3098_v2 (NCBI Assembly GCA_022788535.1) of the hexaploid cultivated hulled oat variety "OT3098," high-throughput resequencing was used to obtain whole-genome sequence information. Whole-genome sequence alignment was then performed using a bioinformatics analysis pipeline to identify and screen for InDel sites. The screening criteria prioritized InDel sites with variant lengths between 100 and 800 bp for subsequent primer design. Variants within this length range offer multiple advantages: moderate variation, enhancing polymorphism identification, and amenability to conventional PCR amplification, ensuring high amplification efficiency and resolution, contributing to the efficiency and stability of subsequent experimental detection.

[0020] Primer design: TBtools software was used for systematic design, with the following key parameters set: 1) PCR product length was controlled between 200 and 600 bp to ensure high band resolution and clear visualization during agarose gel electrophoresis; 2) primer annealing temperature (Tm) was limited to 52–62°C to ensure a balance between specificity and efficiency of the amplification reaction; and 3) primer length was set between 18 and 30 bp to enhance specific binding to the template. All designed primers were validated through secondary structure prediction and specificity verification to avoid interference from primer dimers or nonspecific amplification.

[0021] After multiple rounds of bioinformatics screening and experimental verification, an InDel molecular marker locus interval that can significantly distinguish between hulled and naked oats was successfully screened out. The locus interval is located in the 610439484-610440026 interval of chromosome 5C of the hulled oat variety OT3098 reference genome Oat_OT3098_v2 (NCBI Assembly GCA_022788535.1) (the nucleotide sequence of this locus interval is shown in SEQ ID NO.5). Based on this locus interval, an InDel molecular marker was designed and named OA-InDel-133. The molecular marker OA-InDel-133 includes a 314-bp deletion variant (nucleotide sequence shown in SEQ ID NO.1) and a 2-bp insertion variant (nucleotide sequence: 5'-AA-3"). A stable 312-bp length fragment difference exists between hulled and naked oats.

[0022] A highly specific primer pair was also selected from the designed primers: forward primer F: 5'-CAACTTAACTTCTATGTTGAC-3' (SEQ ID NO. 2) and reverse primer R: 5'-CAAACTAGCTTTTCTAACAA-3' (SEQ ID NO. 3). These primers effectively amplify the target InDel region located on chromosome 5C of Oat husk, with an expected amplified product length of 231 bp. Experimental validation demonstrated that this primer pair consistently amplified a 231 bp specific band in Oat husk samples, consistent with the theoretical target fragment length. In naked oats, however, a 543 bp major band or no amplified band was amplified, demonstrating strong discrimination and good amplification specificity.

[0023] Example 2: Identification of the effectiveness of InDel molecular markers and primer pairs The following method was used to identify the germplasm of 48 oat materials with known skin / naked traits to verify the accuracy of the InDel molecular markers and primer pairs screened in Example 1. The information of the oat materials used in this example is shown in Table 1 below.

[0024] Table 1.48 oat material information

[0025] The identification method is as follows: (1) Genomic DNA extraction: Culture oat materials in a petri dish. When the plants grow to the true leaf stage, collect the leaves and cut them into pieces and place them in a 2.0 mL grinding tube. Add two stainless steel grinding beads and freeze them in liquid nitrogen. Grind them at 70 Hz for 5 minutes. Repeat this operation 3-5 times. After grinding, add 1 mL of CTAB lysis buffer, mix thoroughly, and place them in a 60℃ water bath for 40 minutes. After incubation, centrifuge at 12000 r / min for 10 minutes. Take about 0.5-2 mL of the supernatant and transfer it to a centrifuge tube. Then add an equal volume of chloroform-isoamyl alcohol mixture (volume ratio: chloroform:isoamyl alcohol = 24:1) to the supernatant. Mix thoroughly and centrifuge again at 12000 r / min for 10 minutes. Then, use a pipette to transfer about 400 μL of the supernatant to a 1.5 mL centrifuge tube. Add an equal volume of isopropanol and store at -20°C for 20-40 minutes. After freezing, centrifuge the supernatant at 12,000 rpm at 4°C for 10 minutes. Discard the supernatant and wash the DNA pellet three times with 75% ethanol and once with absolute ethanol. Dry at room temperature for 1 hour. Dissolve the DNA in TE solution or ddH2O. Once fully dissolved, store at -20°C until ready for use.

[0026] (2) PCR amplification: The DNA sample obtained in step (1) was amplified by PCR using a designed and synthesized specific primer pair (forward primer F: 5'-CAACTTAACTTCTATGTTGAC-3' (SEQ ID NO. 2) and reverse primer R: 5'-CAAACTAGCTTTTCTAACAA-3' (SEQ ID NO. 3)). The total reaction volume was 20 µL, specifically including: 0.5 µL genomic DNA template, 10 µL 2× Rapid Taq Master Mix, 1 µL forward primer, 1 µL reverse primer, and ddH2O to 20 µL (i.e., 7.5 µL). Amplification was performed using a touchdown PCR protocol with the following reaction conditions: initial denaturation at 94°C for 5 minutes, followed by a two-stage cyclic amplification: the first stage consisted of 10 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 25 seconds, and extension at 72°C for 25 seconds. The second stage consisted of 30 cycles of denaturation at 94°C for 25 seconds, followed by a 0.5°C decrease in annealing temperature from 58°C to 53°C, and extension at 72°C for 25 seconds. A final extension at 72°C for 5 minutes was performed after the amplification was completed. The resulting amplified product was used for subsequent analysis.

[0027] (3) Detection of PCR amplification products: Weigh 4.0 g of agarose and add it to 100 mL of 1× TAE electrophoresis buffer. Heat it in a microwave oven and dissolve it thoroughly to prepare a 4% agarose gel. After the temperature drops slightly, add 10 µL of ethidium bromide (EtBr) nucleic acid dye and mix thoroughly. Place the electrophoresis comb in the gel mold, pour the gel and cool it naturally until it is completely solidified. Then add an appropriate amount of 1× TAE buffer to the electrophoresis tank to completely immerse the gel. Take 10 µL of PCR amplification product (taken from step (2)) and 10 µL of DNA sample (control sample marker, taken from step (1), marked as M in the electrophoresis diagram) and add them to the gel sample wells respectively. Connect the electrodes to the electrophoresis instrument and set a constant voltage of 130 V for electrophoresis until the amplified bands are fully separated.

[0028] 48 oat materials were identified by the above method, and the agarose gel electrophoresis results showed that Figure 1 and Figure 2 : For oats, due to the presence of the InDel site interval in the target region, the oats sample has a 2 bp insertion at sites 106-107 and a 314 bp deletion at sites 212-525. Therefore, a specific band of 231 bp can be amplified, and a single clear band appears in the 200-300 bp lane in the electrophoresis diagram; for naked oats, if the R-terminal primer binds successfully, since there is no InDel site interval, and the length of the InDel site interval is 543 bp, the amplified product is 543 bp long, and a band can be observed in the 500-600 bp position in the electrophoresis diagram; if the R-terminal primer cannot match, the PCR amplification fails, and no characteristic band is displayed in the electrophoresis diagram.

[0029] To verify the specificity of the PCR amplification product, this example also performed cloning and sequencing analysis of the target band. The specific steps were as follows: First, the approximately 458 bp target amplified band was excised and purified using the sanPrep column-based DNA gel extraction kit. The purified product was then ligated with the pEA sY-T1 cloning vector (Beijing Quanshijin Biotechnology Co., Ltd.). The ligation system consisted of 4 µL of the recovered product, 1 µL of the T vector, and 5 µL of solution I, and the reaction was incubated at 16°C for 2 hours. The ligation product was transformed into DH5α competent cells and cultured on plates. Positive clones were screened by PCR and selected for inoculation into LB liquid medium, incubated at 37°C and 220 rpm with shaking for 12 hours. Plasmid DNA from the positive clones was extracted and Sanger sequencing was performed using the T7 universal primer (provided by Beijing Qingke Biotechnology Co., Ltd.). Sequencing analysis using DNAMAN software revealed that the amplified region of the naked oat sample (see SEQ ID NO. 6) was highly consistent with the sequence of the InDel molecular marker locus (SEQ ID NO. 5) (chromosome 5C positions 610439484–610440026 of the Oat OT3098 reference genome), while the amplified sequence of the Oat hull sample (SEQ ID NO. 4) only partially matched this region (displaying significant differences from the reference genome). This result confirms the specificity of the InDel marker and provides a reliable molecular genetic basis for oat species identification.

[0030] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An InDel molecular marker for oat bran, characterized by: The molecular markers are a group of 314 bp deletion variants and 2 bp insertion variants located in the 610439484-610440026 interval of chromosome 5C of the reference genome Oat_OT3098_v2 (NCBI Assembly GCA_022788535.1) of the oat variety OT3098. The nucleotide sequence of the deletion variant is shown in SEQ ID NO.1, and the nucleotide sequence of the insertion variant is 5'-AA-3'.

2. The InDel molecular marker according to claim 1, wherein: Also included is a primer pair for detecting the molecular marker, wherein the primer pair includes a forward primer as shown in SEQ ID NO.2 and a reverse primer as shown in SEQ ID NO.

3.

3. A method for detecting the InDel molecular marker according to claim 1 or 2, characterized in that: PCR amplification is performed using the primer pair described in claim 2, and the molecular marker amplification product is identified by electrophoresis as a 231 bp band on an agarose gel.

4. The detection method according to claim 3, wherein: The nucleotide sequence of the 231 bp band is shown in SEQ ID NO.4, in which bases Y, S, R, M, and K are non-conservative bases.

5. The detection method according to claim 3, wherein: The PCR amplification system comprises 0.2 to 1.0 μL of DNA template, 10 μL of 2×Taq Master Mix, 1 μL of the forward primer described in claim 2, 1 μL of the reverse primer described in claim 2, and the balance is ddH2O per 20 μL.

6. The detection method according to claim 3, wherein: The PCR amplification procedure was a touchdown PCR procedure, and the program reaction conditions were: pre-denaturation at 94-95°C for 3-5 min; followed by two-stage cycling: the first stage was denaturation at 94-95°C for 15-40 s, annealing at 55-62°C for 15-40 s, and extension at 72°C for 15-40 s, for a total of 6-12 cycles; The second stage was denaturation at 94-95°C for 15-40 s, annealing at 50-55°C for 15-40 s, and extension at 72°C for 15-40 s, for a total of 25-35 cycles; finally, extension at 72°C for 5 min and storage at 4°C.

7. Use of the InDel molecular marker according to claim 1 or 2 or the detection method according to any one of claims 3 to 6 in identifying oat germplasm resources.

8. The use according to claim 7, characterized in that: The method for identifying oat germplasm resources comprises extracting genomic DNA of the test object as a template, performing PCR amplification and electrophoresis identification using the primer pair described in claim 2, and observing the electrophoresis results. The oats showing a 200-300 bp band in the electrophoresis lane are hulled oats, and the oats showing a 500-600 bp band or no characteristic band are naked oats.

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