Transposon of tomato male sterility gene SlMs10 as well as InDel molecular marker and application of transposon

Abstract

The invention discloses a transposon of a tomato male sterility gene SlMs10 as well as an InDel molecular marker and application of the transposon. The sequence of the transposon is as shown in SEQ ID NO.3, an InDel molecular marker is developed based on the transposon, the InDel molecular marker is specifically ms10IN4874, and the nucleotide sequence of the InDel molecular marker comprises the sequence as shown in SEQ ID NO.1 and the sequence as shown in SEQ ID NO.2. The invention further designs a primer group for amplifying the molecular marker, the sequence of an upstream primer is as shown in SEQ ID NO.4, and the sequence of a downstream primer is as shown in SEQ ID NO.5. The molecular marker is completely based on the tomato male sterility gene SlMs10, is used for molecular marker-assisted selection of tomato male sterility, and can greatly improve the selection accuracy and shorten the breeding period, thereby accelerating the breeding process.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and particularly relates to a tomato male sterility gene SlMs10 Transposons and their InDel molecular markers and applications. Background Art

[0002] Tomato is a self-pollinating plant with significant hybrid advantages. Compared with ordinary varieties, hybrid varieties have the characteristics of high yield, vigorous growth, strong adaptability, high uniformity and strong stress resistance (Zhou Changjiu et al., 1996). At present, tomato hybrid seed production mostly adopts artificial emasculation and pollination, which is time-consuming, labor-intensive and has high seed production costs; and the purity of hybrids may be affected due to untimely or incomplete emasculation (WANG H, et al., 2018).

[0003] Plant male sterility is common in the plant kingdom. Male sterile plants have abnormal stamen development. Using male sterile lines to produce seeds can simplify the seed production process, save labor, and improve seed purity. Previous researchers have classified and located more than 55 sterility genes on various chromosomes through the identification and gene mapping of various sterile mutants of tomatoes (Xing Hucheng et al., 2004). ms15 , ms26 It is located on chromosome 2. ms3 , ms3 2 and ms42 The meiotic abortion is located on chromosome 2. ms10 and those located on chromosomes 2 and 4 ms5 , ps-2 Genes and tetrad period ms4 Located on chromosome 4 (Bistra. 1999), aborted at the microspore stage ms9 Several genes have also been located. se5.1 It was located on chromosome 5 (Li Yuanlong et al., 2016). sl-2 It is located on chromosome 6, and the remaining ms genes belong to chromosomes 3, 4, 6, 8, 10, and 11 respectively (Zhou Tao et al., 2022).

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

[0005] The object of the present invention is to provide a tomato male sterility gene SlMs10Transposon and InDel molecular marker and application thereof. The present invention discovered the male sterile mutant P220-ms in the natural growth of the tomato high-generation inbred line P220. By comparing and analyzing the growth and development morphology and fertility-related gene expression levels of the male sterile mutant, the first-generation sequencing verified that the mutant gene was SlMs10 , and subsequently discovered the transposon on this gene, and developed a gene-based SlMs10 The InDel marker has important theoretical and practical significance for screening male sterile lines for tomato hybrid breeding.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The invention provides a tomato male sterility gene SlMs10 The transposon has a nucleotide sequence as shown in SEQ ID NO.3.

[0007] The invention also provides application of the transposon in cultivating male sterile tomato varieties.

[0008] Furthermore, by inserting the transposon into the tomato genome, the tomato ms10 Loss of gene function results in male sterile tomato varieties.

[0009] The present invention also provides a tomato male sterility gene SlMs10 The InDel molecular marker is developed based on the transposon, specifically ms10IN4874, and its nucleotide sequence includes the sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO.2.

[0010] The present invention also provides a pair of primer sets, which are the amplification primers for the molecular marker, and include 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.

[0011] The present invention also provides a PCR amplification kit, which comprises the primer set and reagents required for amplification.

[0012] Furthermore, the reagents required for the amplification include Taq DNA polymerase, PCR buffer and dNTPs.

[0013] The present invention also provides the use of the molecular marker, the primer set or the PCR amplification kit in detecting different tomato germplasm resources.

[0014] The present invention also provides a method for screening male sterile tomatoes, which specifically comprises the following steps: extracting genomic DNA of a tomato to be tested, and using the genomic DNA as a template, performing PCR amplification on the template using the primer set or the PCR amplification kit, performing electrophoresis on the amplified product, and determining the type of the tomato based on the amplified product; if the amplified product is 500bp-750bp, the tomato to be tested is determined to be a wild-type tomato, and if the amplified product is greater than 5000bp, the tomato to be tested is determined to be a male sterile tomato.

[0015] Furthermore, the conditions for PCR amplification are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 5 min, 35 cycles; extension at 72°C for 10 min; and storage temperature at 4°C.

[0016] Furthermore, the PCR reaction system is: 0.5 μL of each primer with a concentration of 10 μmol / L, 5 μL of 2×MasterMix, 2 μL of a DNA template with a concentration of 30-50 ng / μL, and ddH2O to make up to 10 μL.

[0017] Further, if the amplified product is a fragment with a length of 528 bp as shown in SEQ ID NO.1, the tomato to be tested is judged to be a wild-type tomato; if the amplified product is a fragment with a length of 5402 bp as shown in SEQ ID NO.2, the tomato to be tested is judged to be a male sterile tomato.

[0018] The present invention also provides the use of the molecular marker, the primer set or the PCR amplification kit in assisting the cultivation of male sterile tomato varieties.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: Based on the analysis of gene expression of tomato male sterility mutation and first-generation sequencing, the present invention successfully identified the tomato male sterility gene SlMs10 The mutation type and the tomato male sterility gene SlMs10 The InDel molecular marker ms10IN4874 was identified and its function was verified. The InDel molecular marker can play an important role in creating male sterile tomato materials and lay the foundation for establishing a molecular marker-assisted selection system for male sterile materials. The present invention can overcome the defect of low efficiency of phenotypic identification of male sterility (recessive traits), thereby efficiently and accurately identifying SlMs10 genotypes to improve the efficiency of screening sterile lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Comparison of flower morphology, pollen viability and fruit morphology between P220 and P220-ms tomatoes.

[0021] Figure 2 for SlMs10 Gene-specific molecular markers.

[0022] Figure 3 P220 and P220-ms SlMs10 Gene expression analysis.

[0023] Figure 4 P220 and P220-ms SlMs10 Agarose gel electrophoresis of gene amplification.

[0024] Figure 5 The F1 generation of the hybrid of P220 and P220-ms SlMs10 Agarose gel electrophoresis of gene amplification.

[0025] Figure 6 This is a co-segregation detection diagram for separated groups.

[0026] Figure 7 For SlMs10 Single-target CRISPR / Cas9 gene editing vector.

[0027] Figure 8 for SlMs10 Schematic diagram of the gene sequence.

[0028] Fig. 9 This is the sequencing result of the Slms10-3 target site.

[0029] Fig.10 Comparison of flower morphology, pollen viability and fruit morphology between tomato gene-edited plants (Slms10-3) and WT (AC).

[0030] Fig.11 For SlMs10 pBI121 gene overexpression vector.

[0031] Fig.12 Comparison of flower morphology, pollen viability and fruit morphology between tomato gene overexpression plants (OE-6) and P220. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0033] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0034] The inventors first analyzed the fruit morphology and pollen viability characteristics of the tomato male sterile mutant and its wild type P220, and then found the mutant through sequencing data analysis. Slms10 The gene had a 4874bp insertion in the 5'UTR, which resulted in loss of gene function, and the molecular marker ms10IN4874 was developed. Slms10 Functional verification was performed using gene editing technology, further confirming that the molecular marker was Slms10 Gene-specific markers, thus laying the foundation for the use of this marker in hybrid breeding using tomato male sterile lines.

[0035] Example 1: Observation of P220 and P220-ms phenotypes The phenotypes of tomato P220 (advanced tomato inbred line) and tomato natural mutant P220-ms were observed. Figure 1 As shown, the stigma of the pistil of the mutant P220-ms plant is prominent, the stamens cannot produce viable pollen, and there are no seeds in the mature fruit, while the stigma of the pistil of the P220 plant is shorter than the stamens, the stamens produce viable pollen normally, and the seeds in the fruit can develop normally.

[0036] Example 2: P220 and P220-ms Slms10 Gene expression analysis The only significant difference between the tomato mutant strain and the wild-type strain was the development of floral organs. The DNA of P220 and mutant P220-ms was used as templates. The PCR reaction system was: 0.5 μL of each primer 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 10 μL. The conditions for PCR amplification were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 5 min, 35 cycles; extension at 72°C for 10 min; storage temperature 4°C. Genes related to floral organ development were amplified. The results of agarose gel electrophoresis are shown in the figure. Figure 2 It was shown that the mutant P220-ms DNA was used as a template SlMs10 The amplified product was significantly larger than the wild type.

[0037] Then, the cDNA of P220 and mutant P220-ms was used as template to analyze the expression of genes related to floral organ development in P220 and P220-ms. Figure 3 It was not detected in the floral organs of the mutant P220-ms SlMs10 expression.

[0038] Example 3: InDel discovery and marker development In the mutant ms10 SlMs10 Mutation analysis was performed using DNA from P220 and mutant P220-ms as templates. SlMs10 Gene-specific primers were designed for amplification and sequencing. Agarose gel electrophoresis results showed that the amplified product of P220 was between 500bp and 750bp, and the amplified product of the mutant P220-ms was greater than 5000bp.

[0039] P220 and mutant P220-ms SlMs10 The amplified products were sequenced. Figure 2 As shown, the mutant SlMs10 There is a 4874bp insertion in the 5'UTR region of the gene (the sequence is shown in SEQ ID NO.3). The sequencing results show that the inserted base is a repetitive retrotransposon, and a gene-specific InDel molecular marker was developed for this.

[0040] The InDel molecular marker was named ms10IN4874, and its nucleotide sequence consisted of the sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO.2.

[0041] A pair of PCR primers ms10IN4874F / R was designed for marker ms10IN4874, including upstream primer Primer1 and downstream primer Primer2. The primer sequences are as follows: Primer1: 5'-CTTCCTTTTATCTGTTCTTCTTCC-3' (SEQ ID NO.4); Primer2: 5'-CTGACCATATTTCCCGTTTG-3' (SEQ ID NO. 5).

[0042] Theoretically, this pair of primers can amplify a fragment with a length of 5402 bp as shown in SEQ ID NO.2 in the genomic DNA of male sterile mutant P220-ms tomato, and a fragment with a length of 528 bp as shown in SEQ ID NO.1 in the genomic DNA of P220 tomato ( Figure 4 ). The male sterile mutant P220-ms tomato was used as the female parent and hybridized with P220 tomato. SlMs10 The genotype of the F1 generation of tomatoes is heterozygous. Using the genomic DNA of the F1 generation of tomatoes as a template, Primer1 and Primer2 primers can amplify heterozygous fragments of 5402 bp and 528 bp in length ( Figure 5 ), the F1 generation tomato was obtained by backcrossing with the ms10 mutant SlMs10 The genotype of F2 generation tomatoes is heterozygous fertile and recessive homozygous male sterile, and the genomic DNA of F2 generation fertile tomatoes is used as a template, such as Figure 6Lanes 11-20, Primer1 and Primer2 primers can amplify hybrid fragments of 5402 bp and 528 bp in length, using F2 generation male sterile tomato genomic DNA as template, e.g. Figure 6 In lanes 1-10, Primer1 and Primer2 primers can amplify a recessive homozygous fragment of 5402 bp in length.

[0043] Embodiment 4: SlMs10 Gene knockouts show phenotypes similar to mutants 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 ), wild-type tomato material AC was stably genetically transformed by Agrobacterium GV3101-mediated method. Finally, 13 transgenic positive plants were obtained. The target editing status of each single plant was detected and analyzed. The results are as follows Figure 8 and Fig. 9 As shown, the plant Slms10-3 had a homozygous mutation with a 1 bp insertion at the target site.

[0044] The floral organs, fruit phenotypes and pollen viability of the wild type WT (AC) and the Slms10-3 mutant were identified. Fig.10 As shown, WT (AC) has normal flower organ development, normal pollen development in stamens, and normal seeds development in fruits, while the stigma of the SlMs10-3 mutant is prominent, the stamens are split and cannot produce viable pollen, and the seeds in the fruit cannot develop normally. The above shows that the phenotype of Slms10-3 is similar to that of the mutant ms10.

[0045] Example 5: Complementation of the natural mutant P220-ms SlMs10 Gene To further verify SlMs10 The function of the gene is investigated in the male sterile mutant P220-ms tomato. SlMs10 Gene transformation was first constructed SlMs10 Ms10-pBI121 overexpression vector ( Fig.11 ). The natural mutant P220-ms was stably transformed by Agrobacterium GV3101-mediated method. Finally, 21 transgenic positive plants were obtained. The overexpression strain OE-6 was selected for subsequent experiments.

[0046] The floral organs, fruit phenotypes and pollen viability of P220 tomatoes and the overexpression line OE-6 were identified. Fig.12As shown, the floral organs of OE-6 developed normally, the pollen in the stamens developed normally, the seeds in the fruit developed normally, and the phenotype and male fertility of the overexpression strain were similar to those of P220 tomatoes.

[0047] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A tomato male sterility gene SlMs10 A transposon, characterized in that The nucleotide sequence of the transposon is shown in SEQ ID NO.

3.

2. Use of the transposon according to claim 1 in breeding male sterile tomato varieties.

3. The use according to claim 2, characterized in that: By inserting the transposon into the tomato genome, the tomato ms10 Loss of gene function results in male sterile tomato varieties.

4. A tomato male sterility gene SlMs10 The InDel molecular marker is characterized by: The molecular marker is developed based on the transposon according to claim 1, specifically ms10IN4874, and its nucleotide sequence includes the sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO.

2.

5. A pair of primer sets, characterized in that: The primer set is the amplification primer for the molecular marker according to claim 4, which comprises 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.

6. A PCR amplification kit, characterized in that: The kit comprises the primer set according to claim 5 and reagents required for amplification.

7. Use of the molecular marker according to claim 4, the primer set according to claim 5 or the PCR amplification kit according to claim 6 in detecting different tomato germplasm resources.

8. A method for screening male sterile tomatoes, characterized in that: The specific steps are: extracting the genomic DNA of the tomato to be tested, and using it as a template, performing PCR amplification on the template using the primer set described in claim 5 or the PCR amplification kit described in claim 6, performing electrophoresis on the amplified product, and judging the type of the tomato based on the amplified product; if the amplified product is 500bp-750bp, the tomato to be tested is judged to be a wild-type tomato, and if the amplified product is greater than 5000bp, the tomato to be tested is judged to be a male sterile tomato.

9. The method according to claim 8, characterized in that The conditions for the PCR amplification are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 5 min, 35 cycles; extension at 72°C for 10 min; and storage temperature at 4°C.

10. Use of the molecular marker according to claim 4, the primer set according to claim 5 or the PCR amplification kit according to claim 6 in assisting the breeding of male sterile tomato varieties.

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