Transposon of tomato male sterility gene slms10, in del molecular marker and application thereof

Abstract

This invention discloses a tomato male sterility gene. SlMs10 The invention relates to a transposon, its InDel molecular marker, and its applications. The sequence of the transposon is shown in SEQ ID NO.3, and an InDel molecular marker, ms10IN4874, was developed based on this transposon. Its nucleotide sequence includes the sequences shown in SEQ ID NO.1 and SEQ ID NO.2. The invention also designs a primer set for amplifying the molecular marker; the sequence of the upstream primer is shown in SEQ ID NO.4, and the sequence of the downstream primer is shown in SEQ ID NO.5. The molecular marker of this invention is entirely based on the tomato male sterility gene. SlMs10 Its use in molecular marker-assisted selection for male sterility in tomatoes can significantly improve the accuracy of selection, shorten the breeding cycle, and thus accelerate the breeding process.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, and particularly relates to a tomato male sterility gene. SlMs10 Transposons and their InDel molecular labeling and applications. Background Technology

[0002] Tomato is a self-pollinating plant with significant hybrid vigor. Compared with ordinary varieties, hybrid varieties have characteristics such as high yield, vigorous growth, strong adaptability, high uniformity, and strong resistance to adverse conditions (Zhou Changjiu et al., 1996). Currently, the production of tomato hybrid seeds mostly adopts manual emasculation and pollination, which is time-consuming, labor-intensive, and costly; moreover, untimely or incomplete emasculation may affect the purity of the hybrid (WANG H, et al., 2018).

[0003] Male sterility is prevalent in the plant kingdom, and male-sterile plants exhibit abnormal stamen development. Using male-sterile lines for seed production can simplify the process, save labor, and improve seed purity. Previous researchers have identified and mapped over 55 male sterility genes to various tomato male sterility mutants onto different chromosomes (Xing Hucheng et al., 2004). For example, genes that cause male sterility during prophase I of meiosis... ms15 , ms26 Located on chromosome 2, ms3 , ms3 2 and ms42 Meiotic abortion is located on chromosome 1, while meiotic abortion is located on chromosome 2. ms10 and simultaneously located on chromosomes 2 and 4 ms5 , PS-2 Genes and tetrad stage ms4 Located on chromosome 4 (Bistra. 1999), aborted during the microspore stage. ms9 Several genes, including those controlling the length of the tomato style, were also located. se5.1 It is located on chromosome 5 (Li Yuanlong et al., 2016). sl-2 Located on chromosome 6, the remaining ms genes belong to chromosomes 3, 4, 6, 8, 10, and 11 respectively (Zhou et al., 2022).

[0004] Therefore, developing molecular markers based on tomato male sterility genes is of great significance for screening tomato male sterile lines for tomato hybridization breeding. Summary of the Invention

[0005] The purpose of this invention is to provide a tomato male sterility gene. SlMs10Transposons and their InDel molecular markers and applications. This invention discovered a male-sterile mutant, P220-ms, in the natural growth of the high-generation tomato inbred line P220. Through comparative analysis of the growth and development morphology and fertility-related gene expression levels of the male-sterile mutant, first-generation sequencing verified that the mutant gene is... SlMs10 Subsequently, transposons on this gene were discovered, and a gene-based approach was developed. SlMs10 The InDel marker is of significant theoretical and practical importance for screening male-sterile lines for tomato hybridization breeding.

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0007] This invention provides a tomato male sterility gene. SlMs10 The transposon has the nucleotide sequence shown in SEQ ID NO.3.

[0008] The present invention also provides the application of the transposon in the breeding of male-sterile tomato varieties.

[0009] Furthermore, by inserting the transposon into the tomato genome, the tomato... ms10 The loss of gene function results in male-sterile tomato varieties.

[0010] This invention also provides a tomato male sterility gene. SlMs10 The InDel molecular marker, which is developed based on the transposon described above, is specifically ms10IN4874, and its nucleotide sequence includes the sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO.2.

[0011] The present invention also provides a pair of primers, which are amplification primers for the molecular markers, comprising an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5.

[0012] The present invention also provides a PCR amplification kit, which includes the aforementioned primer set and reagents required for amplification.

[0013] Furthermore, the reagents required for the amplification include Taq DNA polymerase, PCR buffer, and dNTPs.

[0014] This invention also provides the application of the aforementioned molecular markers, primer sets, or PCR amplification kits in detecting different tomato germplasm resources.

[0015] The present invention also provides a method for screening male-sterile tomatoes, the specific steps of which are as follows: extracting genomic DNA from the tomato to be tested, and using it as a template, performing PCR amplification on the template using the primer set or the PCR amplification kit, performing electrophoresis on the amplification product, and determining the tomato type based on the amplification product; if the amplification product is 500bp-750bp, the tomato to be tested is determined to be a wild-type tomato, and if the amplification product is greater than 5000bp, the tomato to be tested is determined to be a male-sterile tomato.

[0016] Furthermore, the PCR amplification conditions are as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 5 min, 35 cycles; 72℃ extension for 10 min; storage temperature 4℃.

[0017] Furthermore, the PCR reaction system is as follows: 0.5 μL each of primers with a concentration of 10 μmol / L, 5 μL of 2 × MasterMix, 2 μL of DNA template with a concentration of 30-50 ng / μL, and ddH2O added to a final volume of 10 μL.

[0018] Furthermore, if the amplification product is a 528bp fragment with the sequence shown in SEQ ID NO.1, the tomato to be tested is determined to be a wild-type tomato; if the amplification product is a 5402bp fragment with the sequence shown in SEQ ID NO.2, the tomato to be tested is determined to be a male-sterile tomato.

[0019] This invention also provides the application of the aforementioned molecular markers, primer sets, or PCR amplification kits in the assisted breeding of male-sterile tomato varieties.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] Based on the analysis of the expression level of the male sterility gene in tomato and first-generation sequencing, this invention successfully identified the male sterility gene in tomato. SlMs10 The mutation type was determined, and the gene associated with male sterility in tomatoes was obtained. SlMs10 The InDel molecular marker ms10IN4874 was identified, and its function was verified. This InDel molecular marker plays an important role in creating male-sterile tomato materials, laying the foundation for establishing a molecular marker-assisted selection system for male-sterile materials. This invention overcomes the low efficiency of phenotypic identification of male sterility (a recessive trait), thus enabling efficient and accurate identification. SlMs10 The genotype can be determined to improve the efficiency of screening for sterile lines. Attached Figure Description

[0022] Figure 1Comparison of flower morphology, pollen viability, and fruit morphology between P220 and P220-ms tomatoes.

[0023] Figure 2 for SlMs10 Gene-specific molecular markers.

[0024] Figure 3 For P220 and P220-ms SlMs10 Gene expression level analysis.

[0025] Figure 4 For P220 and P220-ms SlMs10 Gene amplification agarose gel electrophoresis image.

[0026] Figure 5 F1 generation of crosses between P220 and P220-ms SlMs10 Gene amplification agarose gel electrophoresis image.

[0027] Figure 6 This is a detection map of co-separation of the separated population.

[0028] Figure 7 In response to SlMs10 A single-target CRISPR / Cas9 gene editing vector.

[0029] Figure 8 for SlMs10 A schematic diagram of a gene sequence.

[0030] Figure 9 The sequencing results are for the Slms10-3 target.

[0031] Figure 10 Comparison of flower morphology, pollen viability and fruit morphology between gene-edited tomato plants (Slms10-3) and WT (AC) plants.

[0032] Figure 11 In response to SlMs10 pBI121 gene overexpression vector.

[0033] Figure 12 Comparison of flower morphology, pollen viability, and fruit morphology between tomato gene overexpression plants (OE-6) and P220. Detailed Implementation

[0034] The technical solution of the present invention will be further described in detail with reference to the following specific examples.

[0035] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0036] The inventors first analyzed the fruit morphology and pollen viability characteristics of the male-sterile tomato mutant and its wild-type P220, and then discovered the mutant through sequencing data analysis. Slms10 A 4874bp insertion in the 5' UTR of the gene led to loss of gene function, prompting the development of the molecular marker ms10IN4874. Following this, [further details were provided]. Slms10 Functional validation was performed using gene editing technology, further confirming that this molecular marker is... Slms10 Gene-specific markers, thus laying the foundation for their use in hybridization breeding using male-sterile tomato lines.

[0037] Example 1: Observation of P220 and P220-ms phenotypes

[0038] The phenotypes of tomato P220 (a high-generation inbred line of tomato) and the natural mutant tomato P220-ms were observed, and the results are as follows: Figure 1 As shown, the mutant P220-ms plant has a prominent stigma on its pistil, the stamens cannot produce viable pollen, and there are no seeds in the mature fruit. In contrast, the P220 plant has a shorter stigma on its pistil than the stamens, the stamens produce viable pollen normally, and the seeds in the fruit can develop normally.

[0039] Example 2: P220 and P220-ms Slms10 Gene expression analysis

[0040] The only significant difference between the tomato mutant lines and wild-type lines during growth and development was in floral organ development. Using DNA from P220 and the mutant P220-ms as templates, the PCR reaction system was as follows: 0.5 μL each of primers at a concentration of 10 μmol / L, 5 μL of 2 × MasterMix, 2 μL of DNA template at a concentration of 30-50 ng / μL, and ddH2O to a final volume of 10 μL. The PCR amplification conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 5 min, 35 cycles; 72℃ extension for 10 min; storage temperature 4℃. Genes related to floral organ development were amplified. Agarose gel electrophoresis results are shown below. Figure 2 The results showed that DNA from the mutant P220-ms was used as a template. SlMs10 The amplified product was significantly larger than that of the wild type.

[0041] Using cDNA from P220 and the mutant P220-ms as templates, the expression of genes related to floral organ development in P220 and P220-ms was analyzed. qRT-PCR analysis is as follows: Figure 3 The results showed that no [specific marker] was detected in the floral organs of the mutant P220-ms. SlMs10 The expression.

[0042] Example 3: Discovery and Tagging Development of InDel

[0043] In mutant ms10 SlMs10 Mutation analysis was conducted using P220 and mutant P220-ms DNA as templates, respectively. SlMs10 Gene-specific primers were designed for amplification and sequencing. Agarose gel electrophoresis results showed that the amplification product of P220 was between 500bp and 750bp, while the amplification product of the mutant P220-ms was greater than 5000bp.

[0044] For P220 and mutant P220-ms SlMs10 The amplified products were subjected to first-generation sequencing, such as... Figure 2 As shown, in the mutant SlMs10 A 4874bp insertion was found in the 5'UTR region of the gene (sequence shown in SEQ ID NO.3). Sequencing analysis revealed that the inserted bases were repetitive retrotransposons, and a gene-specific InDel molecular marker was developed for this purpose.

[0045] The InDel molecular marker was named ms10IN4874, and its nucleotide sequence consists of sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0046] A pair of PCR primers, ms10IN4874F / R, was designed for the marker ms10IN4874, including upstream primer Primer1 and downstream primer Primer2. The primer sequences are as follows:

[0047] Primer1: 5'-CTTCCTTTTATCTGTTCTTCTTCC-3' (SEQ ID NO.4);

[0048] Primer2: 5'-CTGACCATATTTCCCGTTTG-3' (SEQ ID NO. 5).

[0049] Theoretically, this primer pair can amplify a 5402 bp fragment as shown in SEQ ID NO.2 from the male-sterile mutant P220-ms tomato genomic DNA, and a 528 bp fragment as shown in SEQ ID NO.1 from the P220 tomato genomic DNA. Figure 4 The male-sterile mutant P220-ms tomato was obtained by crossing it with P220 tomato as the female parent. SlMs10 Using F1 generation tomatoes with heterozygous genomic DNA as a template, Primer1 and Primer2 primers can amplify heterozygous fragments of 5402 bp and 528 bp in length, respectively. Figure 5F1 generation tomatoes were obtained by backcrossing with the ms10 mutant. SlMs10 F2 generation tomatoes with genotypes of heterozygous fertile and recessive homozygous male-sterile, using F2 generation fertile tomato genomic DNA as a template, such as... Figure 6 Lanes 11-20: Primer 1 and Primer 2 amplified heterozygous fragments of 5402 bp and 528 bp in length, respectively. Using F2 male-sterile tomato genomic DNA as a template, ... Figure 6 Lanes 1-10: Primer1 and Primer2 can amplify a recessive homozygous fragment of 5402 bp in length.

[0050] Example 4: SlMs10 Gene knockout results in a phenotype similar to that of the mutant.

[0051] To verify SlMs10 The function of genes, in SlMs10 A single target site was designed on the second exon and cloned into the CRISPR / Cas9 gene editing vector pYLCRISPR / Cas9Pubi-H. Figure 7 Stable genetic transformation of wild-type tomato material AC was performed using Agrobacterium GV3101-mediated transformation. A total of 13 transgenic positive plants were obtained. The target site editing status of each individual plant was detected and analyzed, and the results are as follows: Figure 8 and Figure 9 As shown, the plant Slms10-3 has a homozygous mutation with a 1bp insertion at the target site.

[0052] The floral organs, fruit phenotype, and pollen viability of wild-type WT(AC) and Slms10-3 mutants were identified, and the results are as follows: Figure 10 As shown, the WT(AC) mutant exhibits normal floral organ development, normal pollen development in the stamens, and normal seed development in the fruit. In contrast, the SlMs10-3 mutant has a prominent stigma, split stamens that fail to produce viable pollen, and abnormal seed development in the fruit. This demonstrates that the SlMs10-3 phenotype is similar to that of the ms10 mutant.

[0053] Example 5: Complementation of the natural mutant P220-ms SlMs10 Gene

[0054] To further verify SlMs10 The function of the gene, in the male-sterile mutant P220-ms tomato SlMs10 Genetic transformation first involves constructing... SlMs10 Ms10-pBI121 overexpression vector of the gene ( Figure 11The naturally occurring mutant P220-ms was stably transformed using Agrobacterium GV3101-mediated transformation. A total of 21 transgenic positive plants were obtained. The overexpressing line OE-6 was then screened for further experiments.

[0055] The floral organs, fruit phenotype, and pollen viability of P220 tomatoes and the overexpression line OE-6 were identified, and the results are as follows: Figure 12 As shown, the OE-6 flower organs developed normally, the pollen in the stamens developed normally, and the seeds in the fruit developed normally. The overexpression line phenotype and male fertility were similar to those of the P220 tomato.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. 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 the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A male sterility gene in tomatoes SlMs10 The application of transposons in the breeding of male-sterile tomato varieties is characterized by, Insert the transposon with the nucleotide sequence shown in SEQ ID NO.

3. SlMs10 The 5'UTR region of the gene makes tomatoes ms10 Gene function loss results in male-sterile tomato varieties.

2. A male sterility gene in tomatoes SlMs10 The InDel molecular marker, characterized by, The molecular marker was developed based on the transposon described in claim 1, specifically ms10IN4874, whose nucleotide sequence includes the sequence shown in SEQ ID NO. 1 and the sequence shown in SEQ ID NO.

2.

3. A pair of primers, characterized in that, The primer set is the amplification primer of the molecular marker according to claim 2, which includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

5.

4. A PCR amplification kit, characterized in that, The kit includes the primer set as described in claim 3 and the reagents required for amplification.

5. A method for screening male-sterile tomatoes, characterized in that, The specific steps are as follows: extract genomic DNA from the tomato to be tested, and use it as a template to perform PCR amplification on the template using the primer set described in claim 3 or the PCR amplification kit described in claim 4. Perform electrophoresis on the amplification products and determine the tomato species based on the amplification products. If the amplification product is a fragment with a length of 528 bp as shown in SEQ ID NO.1, the tomato to be tested is determined to be a wild-type tomato. If the amplification product is a fragment with a length of 5402 bp as shown in SEQ ID NO.2, the tomato to be tested is determined to be a male-sterile tomato.

6. The method according to claim 5, characterized in that, The PCR amplification conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 5 min, 35 cycles; 72℃ extension for 10 min; storage temperature 4℃.

7. The application of the molecular marker of claim 2, the primer set of claim 3, or the PCR amplification kit of claim 4 in the assisted breeding of male-sterile tomato varieties.

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