A SCAR molecular marker for distinguishing the color of onion bulbs and its application

Abstract

This invention belongs to the field of crop molecular breeding technology, specifically relating to a SCAR molecular marker for distinguishing onion bulb color and its application. The nucleotide sequence of the SCAR molecular marker in purple-red onions is shown in SEQ ID NO:1, and the nucleotide sequence in yellow onions is shown in SEQ ID NO:2. Furthermore, a primer set based on this molecular marker was developed, which can identify onion bulb color through DNA detection at the seedling stage. The results are stable and unaffected by the environment, overcoming the drawback of needing to perform color screening after bulb enlargement in onion breeding, achieving the purpose of early identification. It can not only be used for marker-assisted breeding of onion bulb color traits to accelerate the breeding process, but also for the identification of onion seed purity and germplasm resources, laying the foundation for marker-assisted selection breeding of onions.

Description

Technical Field

[0001] This invention belongs to the field of crop molecular breeding technology, specifically relating to a SCAR molecular marker for distinguishing the color of onion bulbs and its application. Background Technology

[0002] onion( Allium cepa L., also known as round onion or jade onion, is a biennial herbaceous plant belonging to the genus Allium in the Amaryllidaceae family. It is an important vegetable crop, rich in vitamins, dietary fiber, anthocyanins and other nutrients. It can be eaten fresh or processed and is known as the "Queen of Vegetables".

[0003] Onion bulb color is not only a core indicator for variety identification and commercial value, but also closely related to plant stress resistance and bulb nutritional quality. With the development of molecular genetics techniques, researchers have identified multiple quantitative trait loci (QTLs) associated with onion bulb color through genetic mapping. Kim et al. developed PCR-based molecular markers based on mutations in the structural genes DFR and ANS, and performed genotypic analysis on yellow and pink onion segregating populations. Baek et al. used BSA combined with RNA-Seq technology to develop the GST1 marker, which is tightly linked to the recessive white C locus, effectively distinguishing white onions controlled by the C locus. However, these molecular markers are only suitable for screening target alleles in segregating populations. Wang Zhenbao et al. developed a cDNA molecular marker to distinguish purple and yellow onions based on the differential expression of the anthocyanin synthase gene in onions with different skin colors. This marker, developed at the RNA level, has a complex operation process, high cost, and is not convenient for early selection in breeding.

[0004] Therefore, developing novel molecular markers that can accurately distinguish the color of onion bulbs is of great significance for accelerating the molecular marker-assisted breeding process of onion bulb color traits. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the purpose of this invention is to provide a SCAR molecular marker for distinguishing the color of onion bulbs and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a SCAR molecular marker linked to the color trait of onion bulbs, with the nucleotide sequence in purple-skinned onions as shown in SEQ ID NO:1 and the nucleotide sequence in yellow-skinned onions as shown in SEQ ID NO:2.

[0007] This invention uses the purple-red onion RY-20F as the maternal parent and the yellow-skinned double haploid material DH17 as the paternal parent to construct an F2 segregating population. A pooled pool of purple-red and yellow onion bulbs from the F2 segregating population was constructed for extreme color traits. Transcriptome sequencing analysis revealed the gene g450131, whose amplified fragment size differed by approximately 400 bp between the two parents. Based on the differential sequence of g450131 in the yellow-skinned onion DH17 and the purple-red onion RY-20F, a SCAR molecular marker closely linked to the onion bulb color trait was developed and named OCM379. Further verification of the SCAR marker OCM379 in inbred lines of onions with different bulb colors demonstrated its close linkage to the onion bulb color trait, indicating that the specific molecular marker OCM379 provided by this invention can be used to identify onion bulb color.

[0008] A second aspect of the present invention provides a primer set for amplifying the SCAR molecular marker described in the first aspect, the primer set comprising an upstream primer with the nucleotide sequence shown in SEQ ID NO:3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO:4.

[0009] The primer set for amplifying SCAR markers provided by this invention is used to identify the color of onion bulbs. Therefore, the application of the primer set in identifying the color of onion bulbs is within the protection scope of this invention.

[0010] A third aspect of the present invention provides a reagent for identifying the color of onion bulbs, comprising the primer set described in the second aspect.

[0011] Furthermore, the reagents include Taq enzyme, dNTPs, and Mg. 2+ The detection primers are designed using the SCAR molecular marker OCM379 as a target. The detection primers consist of an upstream primer with the nucleotide sequence shown in SEQ ID NO. 3 and two primers with the nucleotide sequence shown in SEQ ID NO. 4.

[0012] A fourth aspect of the present invention provides a kit for identifying the color of onion bulbs, comprising the primer set described in the second aspect or the reagent described in the third aspect.

[0013] A fifth aspect of the invention provides the use of the SCAR molecular marker described in the first aspect, the primer set described in the second aspect, the reagent described in the third aspect, or the kit described in the fourth aspect in any of the following: (1) Detect or identify the color of onion bulbs; (2) Selection of onion bulb color; (3) Onion molecular marker-assisted breeding.

[0014] Furthermore, the marker is used as a molecular marker to detect or identify the color of onion bulbs during the seedling stage via DNA detection. Specifically, the marker is used to perform PCR amplification on the genomic DNA of the sample to be tested using a primer set. The color of the onion bulb is determined based on the length of the PCR amplification product. If the length of the PCR amplification product is only 955 bp, the bulb color of the sample is determined to be purplish-red; if the length of the PCR amplification product is only 554 bp, the bulb color of the sample is determined to be yellow; if the length of the PCR amplification product includes both 955 bp and 554 bp, the bulb color of the sample is determined to be pink.

[0015] Furthermore, the application of the SCAR molecular marker in the selection of onion bulb color includes the following steps: (1) Extract DNA from the sample to be tested; (2) Design upstream and downstream primers using SCAR molecular marker OCM379 as the target for PCR amplification; (3) If the length of the PCR amplification product is only 955 bp, the bulb color of the sample to be tested is judged to be purplish-red; if the length of the PCR amplification product is only 554 bp, the bulb color of the sample to be tested is judged to be yellow; if the length of the PCR amplification product includes both 955 bp and 554 bp, the bulb color of the sample to be tested is judged to be pink.

[0016] In the nucleotide sequence of onion bulb color, the specific SCAR marker of the present invention shows a 955 bp band in the DNA amplification product of homozygous purple-red onions and a 554 bp band in the DNA amplification product of homozygous yellow onions; the DNA amplification product of heterozygous pink onions shows two bands, 955 bp and 554 bp. Therefore, the above-mentioned specific SCAR marker can also be used to detect the homozygosity of the genotype of onion materials regarding bulb color traits. The application of the specific SCAR marker described in the present invention in identifying the purity of the genotype of onion bulb color traits is within the scope of protection of the present invention.

[0017] A sixth aspect of the present invention provides a method for identifying the bulb color of yellow-skinned onions and purplish-red-skinned onions, comprising the following steps: The primer set described in the second aspect is used to perform PCR amplification on the sample to be tested, and the amplified fragments are identified based on their characteristics. The sample to be tested was an onion seedling; The identification method is agarose gel electrophoresis or first-generation sequencing.

[0018] Furthermore, the PCR amplification reaction system includes: DNA polymerase, reaction buffer, amplification primers, DNA of the sample to be tested, and sterile water.

[0019] Furthermore, the PCR amplification reaction conditions are as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ extension for 10 min, and storage at 4℃.

[0020] Furthermore, the identification method is agarose gel electrophoresis. If the electrophoresis result of the sample shows a specific band of 955 bp, the bulb color of the sample is purplish-red; if the electrophoresis result of the sample shows a specific band of 554 bp, the bulb color of the sample is yellow; if the electrophoresis result of the sample shows both 955 bp and 554 bp bands, the bulb color of the sample is pink.

[0021] Furthermore, the identification method is first-generation sequencing. If the sequence of the electrophoretic recovered fragment is as shown in SEQ ID NO:1, the bulb color of the sample to be tested is purplish-red; if the sequence of the electrophoretic recovered fragment is as shown in SEQ ID NO:2, the bulb color of the sample to be tested is yellow; if the sequence of the electrophoretic recovered fragment is as shown in both SEQ ID NO:1 and SEQ ID NO:2, the bulb color of the sample to be tested is pink.

[0022] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention provides a molecular marker closely linked to the color trait of onion bulbs. Using this molecular marker, the color of onion bulbs can be identified through DNA detection during the seedling stage. The results are stable and unaffected by the environment, overcoming the drawback of color screening in onion breeding after bulb enlargement. This achieves the purpose of early identification and can not only be used for molecular marker-assisted breeding of onion bulb color traits to accelerate the breeding process, but also for the identification of onion seed purity and germplasm resources, laying the foundation for molecular marker-assisted selection breeding of onions. Attached Figure Description

[0023] Figure 1 This is a phenotypic diagram of the segregation traits of the F2 population in Example 1 of the present invention.

[0024] Figure 2 This is a schematic diagram of the amplified fragment of the differentially expressed gene g450131 in the parent genome and the total DNA of its F1 generation in Example 1 of the present invention.

[0025] Figure 3 This is a sequence alignment of the differential gene g450131 in the parents RY-20F and DH17 in Example 1 of the present invention.

[0026] Figure 4This is for the verification of the OCM379 marker in the parental RY-20F and DH17 in Example 1, where M represents the DNA molecular weight standard, 1 to 8 represent the amplification of the parental RY-20F, and 9 to 16 represent the amplification of the parental DH17.

[0027] Figure 5 This is for the verification of the OCM379 marker in the F2 segregating population in Example 1. Here, M represents the DNA molecular weight standard, 1 to 10 represent the amplification of purple-skinned onions in the F2 population; 11 to 20 represent the amplification of pink-skinned onions in the F2 population; 21 to 30 represent the amplification of yellow-skinned onions in the F2 population; 31 to 33 represent the amplification of parents RY-20F, DH17, and F1 generation, respectively; and 34 represents the negative control.

[0028] Figure 6 This is for the verification of the OCM379 marker in 45 inbred lines in Example 1, where M represents the DNA molecular weight standard. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 invention pertains.

[0030] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0031] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0032] Experimental materials: Purple-red onion RY-20F, yellow-skinned double haploid material DH17, and onion inbred lines with different bulb colors were obtained from the Onion Research Group of the Vegetable Research Institute of Shandong Academy of Agricultural Sciences. They are available to the public from the Vegetable Research Institute of Shandong Academy of Agricultural Sciences. These biological materials are only used to repeat the relevant experiments of this invention and should not be used for other purposes.

[0033] Example 1: Filtering of Tags 1. Group building Using the purple-red onion RY-20F as the female parent and the yellow-skinned double haploid material DH17 as the male parent, seeds of the F1 generation were obtained after hybridization. The bulbs of the F1 generation were pink. Self-pollination of the F1 generation yielded the segregating F2 generation, from which a total of 341 onion bulbs were harvested. The bulb colors were purple-red, pink, and yellow, with 99 purple-red bulbs, 155 pink bulbs, and 87 yellow bulbs. Figure 1 The chi-square test value was 3.663, indicating that the purplish-red and yellow skin traits in this F2 segregating population conformed to Mendelian inheritance law of 1:2:1, and that the purplish-red trait of the bulb was dominant over the yellow trait.

[0034] 2. Extraction of DNA and RNA The extraction methods for genomic DNA followed the instructions for the Rapid Plant Genome Extraction Kit produced by Beijing Tiangen Biotech Co., Ltd., and the extraction methods for total RNA followed the instructions for the RNAprep Pure Plant Total RNA Extraction Kit produced by Beijing Tiangen Biotech Co., Ltd.

[0035] 3. Obtaining differentially expressed genes Using purple-red and yellow onion bulbs from the F2 segregating population, a pool of extreme skin color traits was constructed, and transcriptome sequencing (RNA-seq) analysis was performed to obtain one differentially expressed gene, g450131.

[0036] The total DNA of the purple-red onion RY-20F and the yellow-skinned double haploid material DH17 was amplified using the full-length primers of this gene. The results showed that the amplified fragment size of g450131 differed by about 400 bp between the two parents. Figure 2 ).

[0037] 4. Specific band recovery, purification, cloning, and sequencing Differential fragment recovery, cloning, sequencing, and DNA sequence alignment analysis revealed that the amplified fragment of g450131 in purple-skinned onions was 1538 bp in size, with its nucleotide sequence shown in SEQ ID NO:5; while the amplified fragment of g450131 in yellow-skinned onions was 1137 bp in size, with its nucleotide sequence shown in SEQ ID NO:6. Figure 3 It can be seen that the amplification size of the g450131 gene in the purple-red onion RY-20F is 401 bases larger than that in the yellow onion DH17. Figure 3 ).

[0038] Amplified fragment of g450131 in RY-20F of purple-skinned onion:

[0039] Amplified fragment of g450131 in yellow onion DH17:

[0040] The recovery of specific bands was performed in accordance with the instructions for use of the Biospin Gel Extraction Kit; molecular cloning of specific bands was performed using the Molecular Cloning II method; DNA sequencing was outsourced to Beijing Qingke Biotechnology Co., Ltd.

[0041] 5. Primer design and synthesis Based on the differential sequence of g450131 in purple-skinned and yellow-skinned onions, primers for the molecular marker OCM379 were designed: forward primer: OCM379-F1: 5'-CGAGCAAATAAACGCCTTCAACGC-3' (SEQ ID NO:3); reverse primer: OCM379-R1: 5'-GGTAGTCCTCCTCTGTTGTTAGTC-3' (SEQ ID NO:4). These primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and purified by PAGE.

[0042] 6. PCR amplification and detection PCR amplification was performed on a Bio-Rad C1000 Touch thermal cycler from Bio-Rad Laboratories, USA. The 25 μL system contained 12.5 μL of 2×Taq PCR Master Mix, 1.0 μL each of Primer 1 and 2 (0.5 μmol / L), 1.0 μL of DNA, and 9.5 μL of ddH2O. The reaction program was 94℃ pre-denaturation for 5 min, [94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min], 35 cycles, 72℃ extension for 10 min, and storage at 4℃. The PCR products were separated and analyzed by electrophoresis on a 1.0% agarose gel, and the gel imaging system was used for automated imaging.

[0043] 7. Validation of the markers in F2 segregating populations and inbred lines Using primers for the DNA molecular marker OCM379, the forward primer: OCM379-F1: 5'-CGAGCAAATAAACGCCTTCAACGC-3' (SEQ ID NO:3) and the reverse primer: OCM379-R1: 5'-GGTAGTCCTCCTCTGTTGTTAGTC-3' (SEQ ID NO:4), PCR verification was performed on the F2 segregating population and inbred lines with known skin colors. The reaction system, amplification procedure, and detection method are as described in "6. PCR Amplification and Detection".

[0044] Using the total genomic DNA from the parents and individual seedlings of the F2 population as templates, PCR amplification was performed using the developed OCM379 molecular marker primers, and the agarose gel electrophoresis results were analyzed. Figure 5As shown, a specific band of 955 bp was amplified in all purple-red onions, a specific band of 554 bp was amplified in yellow onions, and both 955 bp and 554 bp bands were amplified in pink onions. The identification results of the OCM379 molecular marker on the color of onion bulbs in the F2 segregating population were completely consistent with the phenotype, with an accuracy of 100%.

[0045] Purple-red onion-specific DNA fragment: (SEQ ID NO:1).

[0046] Yellow onion-specific DNA fragment: (SEQ ID NO:2).

[0047] Example 2: Applicability verification of the OCM379 marker in onion bulb color identification To verify the applicability of the OCM379 marker in identifying onion bulb color, PCR validation was performed on 45 onion inbred lines with different bulb colors. 1% agarose gel electrophoresis results showed that the OCM379 molecular marker was tightly linked to the purple-red bulb trait in the inbred lines, with an accuracy of 97.8%. Figure 6 (Table 1).

[0048] Table 1. Validation of the OCM379 marker in 45 onion inbred lines with different bulb colors.

[0049] Note: The 45 materials are inbred lines created by the Onion Research Group of the Vegetable Research Institute of Shandong Academy of Agricultural Sciences. Among them, the materials starting with 1 are cytoplasmic male sterile lines, the materials starting with 3 are maintainer lines, and the materials starting with 7 are paternal lines.

[0050] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A SCAR molecular marker linked to the color trait of onion bulbs, characterized in that, The nucleotide sequences in purple-skinned onions are shown in SEQ ID NO:1, and the nucleotide sequences in yellow-skinned onions are shown in SEQ ID NO:

2.

2. The primer set for amplifying the SCAR molecular marker according to claim 1, characterized in that, The primer set consists of an upstream primer with the nucleotide sequence shown in SEQ ID NO:3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO:

4.

3. A reagent for identifying the color of onion bulbs, characterized in that, Includes the primer set as described in claim 2.

4. A kit for identifying the color of onion bulbs, characterized in that, Includes the primer set as described in claim 2 or the reagent as described in claim 3.

5. The use of the SCAR molecular marker of claim 1, the primer set of claim 2, the reagent of claim 3, or the kit of claim 4 in any of the following: (1) Detect or identify the color of onion bulbs; (2) Onion molecular marker-assisted breeding.

6. A method for identifying the bulb color of yellow-skinned onions and purplish-red-skinned onions, characterized in that, Includes the following steps: The primer set described in claim 2 was used to perform PCR amplification on the sample to be tested, and the amplified fragments were identified based on their characteristics. The sample to be tested was an onion seedling; The identification method is agarose gel electrophoresis or first-generation sequencing.

7. The method according to claim 6, characterized in that, The PCR amplification reaction system includes: DNA polymerase, reaction buffer, amplification primers, DNA from the sample to be tested, and sterile water.

8. The method according to claim 6, characterized in that, The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ extension for 10 min, and storage at 4℃.

9. The method according to claim 6, characterized in that, The identification method is agarose gel electrophoresis. If the electrophoresis result of the sample shows a specific band of 955 bp, the bulb color of the sample is purplish-red; if the electrophoresis result of the sample shows a specific band of 554 bp, the bulb color of the sample is yellow; if the electrophoresis result of the sample shows both 955 bp and 554 bp bands, the bulb color of the sample is pink.

10. The method according to claim 6, characterized in that, The identification method is first-generation sequencing. If the sequence of the electrophoretic recovered fragment is as shown in SEQ ID NO:1, the bulb color of the sample to be tested is purplish-red; if the sequence of the electrophoretic recovered fragment is as shown in SEQ ID NO:2, the bulb color of the sample to be tested is yellow; if the sequence of the electrophoretic recovered fragment is as shown in both SEQ ID NO:1 and SEQ ID NO:2, the bulb color of the sample to be tested is pink.

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