Radish based on the function of the vrnl gene bolting character marker and its application

Abstract

The application belongs to the field of vegetable crop genetics and breeding and biotechnology, and provides a radish bolting trait functional marker based on a VRN1 gene and application thereof. InDel sequence difference is identified in a RsVRN1 gene promoter on a chromosome 6 of radish germplasm with extremely significant difference in bolting, and InDel marker primer pair Nau-VF: 5'-GTCAAACATATATGATCTATCTAGACCTTGC-3' and Nau-VR: 5'-GTTGAGAGAGAAACAGTGATGAAGGA-3' is developed. The functional marker based on the VRN1 gene developed in the application can realize rapid and accurate identification of radish plant bolting. The nucleotide fragment amplified from the marker in radish genomic DNA presents a specific band type after agarose gel electrophoresis, can rapidly and accurately identify radish germplasm bolting, is beneficial to efficient radish late bolting trait identification and marker-assisted selection, and accelerates the radish late bolting fine new variety breeding process.

Description

Technical Field

[0001] This invention belongs to the field of vegetable crop genetics and breeding and biotechnology, and relates to the development and application of an InDel marker for bolting traits in radishes. Background Technology

[0002] Radish (Raphanus sativus L.) is an important root vegetable native to my country, playing a vital role in vegetable production and year-round supply. Radishes are vernalization-promoting plants; after low-temperature vernalization treatment, high temperatures and long days promote bolting and flowering. The sustained low temperatures in winter and early spring easily induce vernalization in radish plants, causing them to shift from vegetative growth to reproductive growth under the long-day conditions of spring, thus promoting bolting and flowering—commonly known as premature bolting. Therefore, radishes are prone to bolting in spring, especially in greenhouse cultivation where premature bolting is severe. This shift from vegetative to reproductive growth inhibits the formation and enlargement of the fleshy root, leading to a decrease in the yield and quality of the radish fleshy root, resulting in a loss of commercial value and a continued tight supply of radishes in the spring market. In recent years, my country's greenhouse horticulture industry has developed rapidly, becoming an important component of high-efficiency agriculture. The application of late-bolting varieties is a fundamental measure to overcome premature bolting in winter and spring greenhouse radish production.

[0003] The genetic basis of late bolting in radish is complex. Using high-throughput RNA sequencing, genes related to bolting and flowering traits were identified in radish, revealing numerous differentially expressed genes and key genes controlling bolting and flowering, such as FT, VRN1, CO, SOC1, FLC, and LFY. The VRN1 (VERNALIZATION 1) gene encodes an APETALA1-like MADS box transcription factor and is an important gene mediating the vernalization pathway. It mainly regulates flowering time by working with the VRN2 gene to maintain low-level expression of the bolting-flowering repressor gene FLC. Based on sequence differences in key genes controlling bolting and flowering, such as FLC, a 1627 bp fragment was found inserted in the first intron of the RsFLC2 gene in the late-bolting material 'Ninengo', leading to the development of a co-dominant marker for the late-bolting trait (Wang Q, et al. A naturally occurring insertion in the RsFLC2 gene associated with late-bolting trait in radish (Raphanus sativus L.). Molecular Breed, 2018, 38:137). However, this marker gene originates from the Japanese late-bolting genotype 'Ninengo', and since the bolting trait in radish is regulated by multiple key genes, the molecular mechanisms affecting bolting and flowering may differ among different genotypes. Therefore, more and more efficient markers need to be developed to effectively identify the late-bolting trait in radish and for marker-assisted selection, thereby accelerating the breeding process of superior late-bolting radish varieties. Summary of the Invention

[0004] This invention addresses the shortcomings of low accuracy, long cycle, and high condition requirements in identifying late bolting traits in radishes. It develops a functional marker for the radish bolting trait based on the VRN1 gene and applies it to the rapid and accurate identification of radish bolting characteristics. This molecular marker can rapidly identify late bolting traits in radishes, providing technical support for efficient, accurate, and rapid identification of late bolting traits in radishes, as well as for marker-assisted selection in the breeding process.

[0005] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0006] This invention first identified a highly significant difference in the promoter sequence of the RsVRN1 gene on chromosome 6 of radish germplasm, and then developed an InDel marker primer pair closely linked to radish bolting behavior, the sequence of which is as follows:

[0007] Nau-VF: 5'-GTCAAACATATATGATCTATCTAGACCTTGC-3';

[0008] Nau-VR: 5'-GTTGAGAGAGAAACAGTGATGAAGGA-3'.

[0009] The present invention also provides an InDel molecular marker for identifying the late bolting trait of radish, wherein the InDel molecular marker is obtained by amplification of the InDel primer pair described in the above technical solution.

[0010] This invention also provides the application of the InDel primer pair described in the above technical solution in identifying bolting traits in radishes. It includes the following steps:

[0011] (1) Genomic DNA was extracted from the tender leaves of the radish material to be tested using the modified CTAB-chloroform-isoamyl alcohol method;

[0012] (2) The genomic DNA of the radish material to be tested was amplified by PCR using the InDel primer pair described in the above technical solution;

[0013] (3) Perform 1.2% agarose gel electrophoresis on the amplification products obtained in step (2), electrophoresis at 150V for 20 min, take a picture under a UV gel imaging device, and observe the amplification results;

[0014] If a single band of 1474 bp is detected in the amplification product, the radish genotype being tested has the late bolting trait.

[0015] If a single band of 827 bp is detected in the amplification product, the radish genotype being tested has the early bolting trait.

[0016] If a double band of 1474bp and 827bp is detected in the amplification product, the radish genotype being tested is heterozygous at this locus, and its bolting tendency is moderate.

[0017] The preferred PCR reaction system is a 10 μL system comprising 10× PCR Buffer (containing Mg). 2+ 1.0 μL of dNTPs (10 μmol / L), 0.2 μL of InDel-F and InDel-R (10 μmol / L) each, 0.5 μL of Taq enzyme (5 U / μL, TaKaRa) 0.1 μL, 1.0 μL of template DNA (10 ng / μL), and ddH2O to a final volume of 10 μL. Mix well and centrifuge.

[0018] The preferred PCR amplification program is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 50 s, 56℃ annealing for 50 s, 72℃ extension for 60 s, 35 cycles; 72℃ extension for 10 min, and finally storage at 4℃.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] This invention is based on the whole genome sequence structure variation analysis of the transcriptome of radish bolting process and the germplasm with extremely significant differences in bolting behavior, which identified the difference in the promoter sequence of the RsVRN1 gene on chromosome 6, and then developed a gene-specific functional marker based on the sequence difference.

[0021] The molecular marker for the major effect site of late bolting of radish provided by this invention has a simple and convenient detection method in practical applications, low requirements for instruments and equipment and technology, clear and simple banding of amplified products, and good stability and product separation effect.

[0022] Breeding and applying bolting-resistant varieties is a fundamental measure to overcome premature bolting in winter and spring greenhouse radish production. This invention is applicable to the identification of bolting traits in radish germplasm. The markers described in this invention can be used to quickly and accurately identify bolting characteristics in radish germplasm during the vegetative growth period, effectively improving the accuracy of bolting trait identification and the efficiency of breeding late-bolting varieties. Attached Figure Description

[0023] Figure 1 The promoter sequences of the very late bolting material 'NAU-LB' and the early bolting material 'Xin li mei' were compared.

[0024] Figure 2 The results of InDel primer amplification in the very late bolting material 'NAU-LB' and the early bolting material 'Xin li mei' are shown. 1, 'NAU-LB'; 2, 'Xin li mei'.

[0025] Figure 3 The results show the amplification of InDel primers in very late and early bolting materials and some individual plants in their F2 population.

[0026] Figure 4 Statistics on bolting time of different band patterns in the F2 population of the very late bolting material 'NAU-LB' and the early bolting material 'NAU-YH' using InDel primers. Detailed Implementation

[0027] The present invention will now be described in conjunction with specific embodiments.

[0028] Example 1

[0029] Obtaining molecular markers for late bolting trait in radish

[0030] This invention utilizes the whole genome sequences of the very late-bolting material 'NAU-LB' and the early-bolting material 'Xin li mei' to detect structural variants (SVs) between the two genomes using bioinformatics methods, with the following parameters:

[0031] The Xin li mei genome was aligned with 'NAU-LB' using the Nucmer program with the parameters "--mum-g1000-c 90-1 40", and sequence filtering was performed using a delta filter with a one-to-one alignment mode (-1). SNPs and InDels were extracted from the one-to-one alignment using the show snp function of the MUMmer4 toolkit with the parameter setting "-ClrH". Functional annotation of SNPs and InDels was performed using the SnpEff package. Detected SVs were functionally annotated using the ANNOVAR package.

[0032] Based on the identified whole-genome alignment results and transcriptome data analysis of radish bolting, SV detection was performed on genes related to late bolting in radish. A 647bp InDel (Insertion-Deletion) marker was found on the promoter of the key radish bolting-resistance gene RsVRN1. Based on the alignment results, an InDel molecular marker closely linked to bolting timing was developed, and its nucleotide sequence is shown in SEQ ID NO: 1. The 647bp insertion sequence is located at bases 293-939 from the 5' end of the nucleotide sequence of the late bolting genotype NAU-LB. (See SEQ ID NO: 1). Figure 1 .

[0033] The following PCR amplification primer pairs were designed for the InDel region mentioned above:

[0034] Nau-VF: 5'-GTCAAACATATATGATCTATCTAGACCTTGC-3' (SEQ ID NO: 2);

[0035] Nau-VR: 5'-GTTGAGAGAGAAACAGTGATGAAGGA-3' (SEQ ID NO: 3).

[0036] Example 2

[0037] Genomic DNA was extracted from the very late bolting material 'NAU-LB' and the early bolting material 'Xin li mei', respectively. Using the genomic DNA as a template, PCR amplification was performed using the InDel primers designed in Example 1 to obtain PCR amplification products. The total volume of the reaction system was 10 μL, including 10×PCR Buffer (containing Mg). 2+1.0 μL of dNTPs (10 μmol / L), 0.2 μL of Nau-VF and Nau-VR (10 μmol / L) each, 0.1 μL of Taq polymerase (5 U / μL, TaKaRa), 1.0 μL of template DNA (10 ng / μL), and ddH2O to a final volume of 10 μL. Mix well and centrifuge. PCR amplification program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 50 s, 56℃ annealing for 50 s, 72℃ extension for 60 s, 35 cycles; 72℃ extension for 7 min, and final storage at 4℃. Detection by 1.2% agarose gel electrophoresis at 150V for 20 min, imaged using a UV gel imaging system. Figure 2 The results showed a significant difference in the length of the amplification products between the two materials. The late-bolting genotype 'NAU-LB' amplified a 1474bp fragment, while the early-bolting genotype 'Xin li mei' could only amplify an 827bp fragment. This verified the accuracy of the target sequence and suggests that it could be further developed as a genetic marker for late-bolting selection in radishes.

[0038] Example 3

[0039] F1 generation was obtained by crossing the very late bolting radish material 'NAU-LB' and the early bolting material 'NAU-YH'. F2 population was constructed through self-pollination, and 104 F2 progeny plants were obtained. Bolting time of the parents and F2 plants was recorded, and genomic DNA was extracted from the parents and all F2 plants using the same extraction method as described above. InDel primers were used to amplify the parents and all F2 plants, and banding patterns were analyzed. The amplification results showed that the very late bolting material 'NAU-LB' amplified a single band of 1474 bp, while the early bolting material 'NAU-YH' amplified a single band of 827 bp. Among the 104 F2 plants, 78 plants amplified a single band of 1474 bp or a double band of 1474 bp and 827 bp, and 26 plants amplified a single band of 827 bp. Figure 3 Based on bolting time statistics, the 26 plants that amplified a single 827bp band exhibited an earlier bolting phenotype than the 78 plants that amplified a single 1474bp band or a double band of 1474bp and 827bp. Figure 4 The above results indicate that the InDel molecular marker described in this invention can rapidly and accurately identify bolting behavior in radishes, and can be applied to the identification of late bolting traits and marker-assisted selection in radishes.

[0040] The above description, in conjunction with specific embodiments, provides a detailed explanation of the present invention, but it should not be construed as limiting the specific implementation of the invention to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the inventive concept, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A functional marker for bolting trait in radish based on the VRN1 gene, characterized in that, The functional marker for the bolting trait of radish is the InDel molecular marker, which is a 647bp insertion / deletion on the promoter of the radish bolting resistance gene RsVRN1; the nucleotide sequence of the InDel molecular marker is shown in SEQ ID No.1, and the nucleotide sequence of the late bolting genotype is a 647bp insertion sequence from the 5' end of the sequence shown in SEQ ID No.1, bases 293-939. The InDel molecular marker was detected using InDel primer pairs. The nucleotide sequence of the upstream primer of the InDel primer pair is shown in SEQ ID No. 2, and the nucleotide sequence of the downstream primer of the InDel primer pair is shown in SEQ ID No.

3. The amplification products were subjected to electrophoresis. If a single band of 1474 bp was detected in the amplification products, the tested radish genotype had the late bolting trait. If a single band of 827 bp was detected in the amplification products, the tested radish genotype had the early bolting trait. If two bands of 1474 bp and 827 bp were detected in the amplification products, the tested radish genotype was heterozygous at that site, and the bolting trait was intermediate.

2. A method for identifying bolting behavior in radish plants, characterized in that, Includes the following steps: (1) Extract genomic DNA from the tender leaves of the radish material to be tested; (2) Perform PCR amplification on the genomic DNA extracted in step (1) using the InDel primer pair described in claim 1; (3) Electrophoresis of amplified products: 1.2% agarose gel electrophoresis detection, 150V electrophoresis for 20 min, photograph under UV gel imaging instrument, and observe amplification results; (4) Identification of bolting trait: If a single band of 1474bp is detected in the amplification product, the radish genotype to be tested has the late bolting trait; if a single band of 827bp is detected in the amplification product, the radish genotype to be tested has the early bolting trait; if a double band of 1474bp and 827bp is detected in the amplification product, the radish genotype to be tested is heterozygous at this locus and has intermediate bolting trait. The radishes mentioned are hybrids of 'NAU-LB', 'Xin li mei', 'NAU-YH', and 'NAU-LB' and 'NAU-YH'.

3. The method according to claim 2, characterized in that, The PCR reaction system described is a 10 μL system containing Mg. 2+ 1.0 μL of 10×PCR Buffer, 0.2 μL of 10 μmol / L dNTPs, 0.5 μL each of 10 μmol / L upstream and downstream primers, 0.1 μL of 5 U / μL Taq enzyme, 1.0 μL of 10 ng / μL template DNA, and ddH2O to a final volume of 10 μL. Mix well and centrifuge.

4. The method according to claim 2, characterized in that, The PCR amplification program is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 50 s, 56℃ annealing for 50 s, 72℃ extension for 60 s, 35 cycles; 72℃ extension for 7 min, and finally storage at 4℃.

5. The application of the InDel primer pair as described in claim 1 in identifying bolting traits in radishes, characterized in that, The method for identifying bolting traits in radishes is as follows: PCR amplification of the radish genomic DNA to be tested is performed using the InDel primers described in claim 1. If a single band of 1474 bp is detected in the amplification product, the radish genotype to be tested has the late bolting trait; if a single band of 827 bp is detected in the amplification product, the radish genotype to be tested has the early bolting trait; if two bands of 1474 bp and 827 bp are detected in the amplification product, the radish genotype to be tested is heterozygous at that locus, and its bolting tendency is intermediate. The radishes mentioned are hybrids of 'NAU-LB', 'Xin li mei', 'NAU-YH', and 'NAU-LB' and 'NAU-YH'.

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