Molecular marker related to tomato anther cracking and application thereof

Abstract

The invention belongs to the technical field of molecular marker-assisted breeding, and particularly relates to a molecular marker related to tomato anther cracking, and the molecular marker is a nucleotide sequence as shown in SEQ ID NO.51. The invention develops an SNP (Single Nucleotide Polymorphism) marker, and the marker can be used for quickly and accurately distinguishing a tomato closed drug type male sterility mutant containing a mutation site, a hybrid plant and a homozygous normal fertility plant. The closed drug type male sterility site is selected through molecular marker assistance, the male sterility material can be rapidly identified in the seedling stage, the labor cost is reduced, and the breeding efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker-assisted breeding technology, specifically relating to a molecular marker related to tomato anther dehiscence and its application. Background Technology

[0002] Tomato is one of my country's important vegetable crops. It is a typical self-pollinating crop with significant hybrid vigor and is characterized by high uniformity and strong resistance to adverse conditions. Currently, tomato hybrid seed production requires artificial emasculation for pollination. However, due to rising labor costs, the use of male-sterile varieties for seed production is becoming a future trend.

[0003] Tomatoes are strictly self-pollinating crops. Their anthers are terminal, forming a closed anther chamber structure through trichomes that lock the anthers together, enclosing the pistil within. Each anther has two locules. During flowering, the anther's inner central line splits longitudinally, allowing mature pollen grains to be released and fall onto the stigma for pollination. Since male sterility was first discovered in tomatoes in 1915, over 50 male-sterile mutants have been reported, all belonging to sporophytic sterility controlled by nuclear genes. Based on the characteristics of the mutant materials, they are mainly divided into three types: sporophytic sterility, structural sterility, and functional sterility. Positional sterility mutants, also known as functional male sterility (Ps), cannot dehisce and release pollen during pollen maturity, thus preventing self-pollination. They can be used as sterile lines or for artificial pollination to propagate sterile lines. Male sterility is widely used in field crops such as corn and rice, but it has not been widely applied in tomatoes. The primary limiting factor for the application of tomato male sterility in hybrid seed production is the difficulty in propagation due to the unique floral structure of tomatoes. Functional male sterility in tomatoes is not true pollen sterility, but rather the production of normal pollen, coupled with abnormal stamen tissue development leading to pollination and fertilization obstacles. A typical example is the style2.1 and exerted(ex) mutants, where the stigma protrudes significantly from the anther tube, preventing mature pollen grains from reaching the stigma after release from the anther, resulting in pollination failure. However, stigma length is easily affected by temperature in the field, exhibiting excessive variation, making it unsuitable for production. Therefore, developing molecular markers closely linked to the tomato anther dehiscence trait is crucial for the rapid and accurate identification of functional male sterility materials during the seedling stage, reducing hybrid seed production costs, and promoting the application of tomato male sterility breeding. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker associated with tomato anther dehiscence, which can be used to rapidly and accurately identify functionally sterile tomatoes during the seedling stage.

[0005] The technical solution adopted in this invention is: This invention provides a molecular marker associated with tomato anther dehiscence, the molecular marker being the nucleotide sequence shown in SEQ ID NO. 51.

[0006] The present invention also provides an application of the molecular marker, wherein the molecular marker is used in any of the following: 1) Screening or assisted screening of male-sterile tomato mutants; 2) To identify or assist in the identification of male-sterile tomato mutants; 3) Tomato hybridization seed production.

[0007] Preferably, the method for identifying male-sterile tomato mutants is as follows: DNA was extracted from the tomatoes to be identified; Using DNA as a template, PCR amplification was performed using the primer sets shown in SEQ ID NO.41 and SEQ ID NO.42, followed by electrophoresis to obtain the PCR product; If the PCR product is sequenced and the genotype is T / T, then the tomato to be identified is male-sterile.

[0008] Preferably, the DNA of the tomato to be identified is derived from tomato leaves.

[0009] Preferably, the PCR amplification system is as follows: 2×M5 HiPer plus Taq HiFi PCR mix 5μL, DNA 1μL, forward primer 0.5μL, reverse primer 0.5μL, ddH2O 3μL.

[0010] Preferably, the PCR amplification procedure is as follows: Pre-denaturation: 95℃ for 3 min, 1 cycle; Denaturation: 94℃ for 25s, annealing at 55℃ for 25s, extension at 72℃ for 15s / 1kb, 35 cycles; Final extension: 72℃ for 5 minutes, 1 cycle.

[0011] Preferably, after electrophoresis, the fertility of the tomato to be identified can be determined based on the band pattern, as follows: If the band size of the tomato to be identified is consistent with that of the ps-like band, then the tomato to be identified is male-sterile.

[0012] A third aspect of the present invention provides a reagent for detecting the fertility of tomatoes, the reagent comprising the primer set as described in claim 3.

[0013] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a molecular marker associated with anther dehiscence in tomato, the molecular marker being the nucleotide sequence shown in SEQ ID NO. 51. This invention also develops an SNP marker that can be used to rapidly and accurately distinguish between tomato anther-closed male-sterile mutants, heterozygous plants, and homozygous fertile normal plants containing the mutant site. This invention utilizes molecular marker-assisted selection of anther-closed male-sterile sites, enabling rapid identification of male-sterile materials at the seedling stage, reducing labor costs, and improving breeding efficiency.

[0014] This invention locates a male sterility gene on chromosome 2 using fine mapping. Sequencing analysis revealed sequence changes in the mutant gene, and based on these sequence changes, a molecular marker was developed. This invention utilizes molecular markers to assist in the selection of anther-closed male sterility sites, enabling rapid identification of male sterile materials at the seedling stage, reducing labor costs and improving breeding efficiency. Furthermore, this invention provides a tomato anther-closed male sterility mutant with a combined anther tube, where petals and anthers are tightly bound, saving the cost of manual emasculation in hybrid seed production. Attached Figure Description

[0015] Figure 1 This study compares the flower morphology, anther morphology, pollen viability, and pollen tube germination of the tomato anther-closed male sterile mutant ps-like and wild currant tomato LA1589. A shows a comparison of flower morphology and petal opening between wild currant tomato and the male sterile mutant; white lines represent 1000 μm. B shows the anther dehiscence in both mutants; white arrows indicate the location of anther dehiscence, and white lines represent 1000 μm. C shows the 0.5% TTC staining of pollen from the sterile parent ps-like and the detection of pollen tube germination ability. D-F show the statistics of the proportion of viable pollen, the proportion of germinating pollen tubes, and the average length of pollen tubes, respectively.

[0016] Figure 2 This section shows the fine mapping of tomato PS-like male sterility mutants with closed-cell anthers. A: Preliminary BSA sequencing results and distribution of InDel markers developed to narrow down candidate regions; B: Fine mapping results and distribution of developed molecular markers, where n represents the population size, P1 is LA1589, P2 is the male sterility mutant PS-like, MF represents fertile recombinant plants, and MS represents sterile recombinant plants; C: Candidate gene coding of PS-like fine mapping candidate regions, where arrows indicate the direction of transcription, and pink squares represent exons of candidate genes. All data is based on the tomato genome annotation information ITAG 4.0 version.

[0017] Figure 3The image shows the bands after polyacrylamide gel staining to narrow down the candidate regions using InDel markers. P1 represents the paternal parent LA1589, P2 represents the maternal parent ps-like, lanes 1-46 represent individual plants from the F2 population, lanes 4, 6, 7, 19, 25, 30, 33, 34, 38, 39, 41, and 46 represent homozygous fertile plants, lanes 5, 8, 13, 28, and 29 represent sterile plants, and double bands represent heterozygous fertile F2 plants.

[0018] Figure 4 The display shows the sequencing results of the sequences amplified using SNP7 specific primers. ps-like is the sterile maternal parent, LA1589 is the fertile paternal parent, F2-31 is a fertile single plant randomly selected from the F2 population, and F2-194 is a sterile single plant randomly selected from the F2 population. Detailed Implementation

[0019] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0020] The inventive concept of this invention is as follows: This invention provides a novel mutant of functional sterility, characterized by closed anthers, environmental stability, and tight adhesion between the anthers and corolla, allowing for manual removal of the corolla. This mutant can be used for seed production of sterile lines and for propagation of sterile lines. The purpose of this invention is to provide molecular markers closely linked to the sterility trait of anther closure for use in assisted breeding.

[0021] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: This invention located the anther dehiscence mutation site in tomato within a 9Mb region on chromosome 2 using BSA pooled sequencing. Further narrowing of the candidate region to 44.5kb using the InDel marker revealed a member of the ABC transporter G family, providing and elucidating a SNP site and molecular marker closely linked to the tomato anther-dehiscence male sterility gene. The physical location of this marker is at base 43,648,064 (SL4.0 version) on tomato chromosome 2. A C base indicates fertility, while a T base indicates sterility. This selected marker has significant theoretical and practical implications for screening male-sterile plants in the seedling stage for tomato hybridization breeding, as detailed below: 1. Phenotypic observation of closed-drug male sterility mutant and wild-type tomato.

[0022] The phenotype and pollen viability of the anther-closed male sterility mutant ps-like and wild currant tomato LA1589 were observed, and the results are as follows: Figure 1 As shown in C.

[0023] Furthermore, pollen viability and pollen tube germination ability of the two materials were measured, proving that the mutant is a functional sterile mutant.

[0024] 2. Development and identification of InDel markers for closed-drug male infertility.

[0025] The male sterile mutant ps-like was selected as the maternal parent and wild currant tomato LA1589 was selected as the paternal parent to construct the F2 population. The sterile pool and fertile pool were constructed by selecting extreme phenotypes and BSA pooled sequencing was performed to locate the region within a 9Mb interval on chromosome 2. InDel markers were designed to narrow down the candidate interval based on the sequencing results.

[0026] 3. Fine localization of the drug-free male infertility gene and acquisition of SNP markers closely linked to the infertility phenotype.

[0027] After narrowing down the candidate interval using InDel markers, InDel markers were further designed within the selected interval to screen recombinant single plants, further narrowing down the candidate interval to within 100kb.

[0028] Further, based on the BSA pooled sequencing results of the sterile population, specific DNA fragments with SNP site polymorphism were screened; through association analysis, SNP sites associated with the tomato drug-free male sterility gene were obtained; using the specific DNA fragments where the SNP sites are located, specific primer pairs for SNP molecular markers were designed to obtain SNP molecular markers closely linked to the tomato drug-free male sterility gene.

[0029] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0030] The list of abbreviations for this invention is shown in Table 1.

[0031] Table 1 List of Abbreviations Example 1 A gene and molecular marker associated with tomato anther dehiscence are as follows: This invention utilizes molecular markers to locate the genes of anther-closed male sterility mutants and obtains SNP molecular markers closely linked to anther closure. Using these SNP molecular markers, it is possible to quickly and accurately distinguish between anther-closed male sterility mutants, heterozygous plants, and homozygous fertile normal plants containing mutation sites.

[0032] 1. Plant materials.

[0033] The tomato male-sterile material ps-like and wild currant tomato LA1589 were both obtained from our laboratory. The anther-closed male-sterile mutant ps-like was a recessive homozygous mutant. After crossing with wild currant tomato LA1589, all F1 offspring were fertile. The F2 population was obtained after self-pollination. All materials were grown at the Shenyang Agricultural University experimental base and managed with routine water and fertilizer.

[0034] 2. Parental phenotype survey.

[0035] During the flowering stage, the open flowers of both parents were observed and photographed. Anther dehiscence was observed under a stereomicroscope, and pollen was stained with 0.5% TTC to compare pollen viability with that of normally open tomato flowers. It was found that the petals and anthers of the anther-closed male-sterile mutant ps-like were tightly joined, forming a closed anther tube that could not dehisce normally. Under a stereomicroscope, the dorsal groove of the anther lobe was observed to be unable to open and release pollen. In contrast, the flowers of the wild currant tomato LA1589 opened normally and were capable of self-pollination. Self-pollination of the male-sterile parents resulted in normal fruit set. Results are shown below. Figure 1 .

[0036] 3. Development of fine localization and molecular markers.

[0037] BSA sequencing was performed using a 42×28 sterile×fertile pool to locate the male sterility gene within a 9Mb region on chromosome 2, between bases 42400001 and 51400000. Based on the sequencing results, InDel-labeled primers (as shown in Table 2) were designed to narrow down the candidate regions. See [link to table]. Figure 2 A.

[0038] Table 2 Primer Information First, DNA was extracted from both parents and individual plants in the F2 population using the CTAB method. After PCR amplification, polyacrylamide gel electrophoresis was performed to observe the genotypic differences between the F2 plants and the two parents. The specific method is as follows: (1) Extraction of plant tissue DNA by CTAB method.

[0039] Take approximately 0.1 g of young plant leaves and grind them into powder in a 2 mL centrifuge tube. Add 700 μL of CTAB solution and invert the mixture 8 times every 10 min for a total of 1 h. Add 500 μL of chloroform-isoamyl alcohol solution (chloroform to isoamyl alcohol volume ratio = 24:1) to the homogenate. Mix thoroughly on a shaker and centrifuge at 12000 rpm for 10 min. After centrifugation, the liquid in the centrifuge tube will separate into three layers. Take approximately 500 μL of the upper supernatant and transfer it to a new 1.5 mL centrifuge tube. Add an equal volume of isopropanol and gently invert the mixture 8 times. Incubate at -20℃ for 30 min. After incubation, centrifuge at 12000 rpm for 10 min and discard the supernatant. Add 1 mL v / v 75% ethanol to wash the precipitate at 12000 rpm. Centrifuge for 5 minutes and discard the supernatant. Centrifuge for another 3 minutes. Use a 100 μL pipette to remove the liquid from the bottom of the tube. Let the tube stand for 15 minutes in a clean bench to allow any residual ethanol to evaporate. Add 50 μL of deionized water and shake until the precipitate dissolves to obtain DNA.

[0040] (2) PCR amplification.

[0041] PCR reactions were performed using Tap HiFi PCR mix, and the amplification system is shown in Table 3.

[0042] Table 3 Amplification System The PCR amplification procedure is shown in Table 4.

[0043] Table 4 PCR Amplification Program In Table 4, "-" indicates that this item is not present.

[0044] (3) Polyacrylamide gel electrophoresis detection.

[0045] An equal volume of bromophenol blue solution was added to the obtained PCR product. The formulation of bromophenol blue solution per 100 mL was: 98 mL deionized formamide, 0.1 g bromophenol blue, 0.1 g xylenenitrile, and 2 mL 0.5 M EDTA·Na (pH=8). Subsequently, a polyacrylamide gel was prepared for detection. Detailed results can be found in [link to results]. Figure 3 The specific method is as follows: First, wipe the flat and concave glass plates with purified water and alcohol respectively, repeating three times. After the glass plates are dry, drop 1 mL of affinity silane onto the flat glass plate and spread it evenly. Spread 1 mL of exfoliating silane evenly onto the concave glass plate, and then wipe them with distilled water and alcohol respectively. Secure and level the plates with fishtail clips. Next, prepare the polyacrylamide gel, pour it into the gel, remove air bubbles, and insert a comb. After standing for 2 hours, the gel will solidify and can be transferred to the electrophoresis tank and placed vertically. After removing the comb, use a flat-head microsyringe to add 3 μL of PCR product with bromophenol blue to the sample well. Set the power to 100W and start electrophoresis until the bromophenol blue dye reaches the bottom of the gel, then stop electrophoresis and begin staining.

[0046] The solutions for each step in the following process are prepared as follows: Fixation: Prepare fixative solution by adding 1L distilled water, 100mL v / v 95% ethanol, and 5mL glacial acetic acid, and fix for 7 minutes.

[0047] Silver staining: 1L distilled water + 1.5g silver nitrate + 2mL formaldehyde, stain for 10min.

[0048] Rinse: Rinse with 1L of distilled water for 2 minutes.

[0049] Staining: 1L distilled water + 11.5g sodium hydroxide + 2mL formaldehyde, shake on a shaker until the bands are clear.

[0050] Preparation of polyacrylamide gel: 80 mL m / m 30% Acrylamide-Bisacrylamide (29:1) + 800 μL m / m 10% APS + 80 μL TEMED.

[0051] Preparation of 8% acrylamide: 270mL m / m 30% Acrylamide-Bisacrylamide (29:1) + 200mL 5×TBE + 530mL distilled water.

[0052] The preparation method for affinity silane working solution is as follows: 25 mL glacial acetic acid + 450 mL anhydrous ethanol + 25 mL affinity silane stock solution; store together with the stripped silane in the dark.

[0053] After staining, the acrylamide gel was observed and the bands were counted. Figure 3 Among them, P1 is the male parent LA1589, P2 is the female parent ps-like, lanes 1 to 46 are individual plants of the F2 population. Those with the same band as the male parent are counted as A, i.e. homozygous fertile individual plants; those with the same band as the female parent are counted as B, i.e. homozygous sterile individual plants; and those with two bands are counted as H, i.e. heterozygous fertile individual plants.

[0054] 4. Genetic analysis and SNP molecular marker development.

[0055] All F1 plants obtained by crossing the anther-closed male-sterile material ps-like as the female parent and wild currant tomato LA1589 as the male parent were fully fertile. Statistical analysis of the F2 population of 767 plants revealed a fertile-to-sterile ratio of approximately 7:1. Existing technologies mention that ps-2 sterile plants are susceptible to environmental and kinship influences; this invention, using a hybrid of cultivated and wild tomatoes, presents factors that may affect the segregation ratio of the offspring.

[0056] The F2 population was used to construct a pool, and the sequencing results of BSA were compared with those of cultivated tomato AC and Heniz 1706. SNP mutation sites that were not repeated in the sequencing of traditional cultivated tomatoes were screened. A total of 10 pairs of SNP markers were designed for genotyping. (See below) Figure 2 B and Table 5.

[0057] Table 5 SNP primer information By designing the InDel marker, the candidate region was narrowed down to a 41.5 kb interval between the Ind4-2 and Ind4-3 markers. Within this candidate region, only the Solyc02g081870 gene has a non-synonymous SNP mutation, namely the SNP7 marker. Genotyping results showed that this molecular marker is tightly linked to the phenotype, with a linkage rate of 93.2%.

[0058] 5. Candidate gene prediction and polymorphic sequence analysis.

[0059] Using the tomato genome annotation information ITAG version 4.0, the fine-mapping region of the male sterility locus encodes six genes. Among them, Solyc02g081870 encodes a gene of the ABCG transporter family. Members of the ABCG transporter family play important roles in the regulation of anther cuticle formation, pollen exine development, callose degradation, and programmed cell death in the anther tapetum. Therefore, Solyc02g081870 is speculated to be a candidate gene regulating tomato dehiscence. See [link to article]. Figure 2 C.

[0060] After PCR amplification, sequencing and sequence alignment were performed. A C / T SNP was found in the coding region of the Solyc02g081870 gene, which differs from that of wild currant tomato LA1589, resulting in a change in one amino acid at the P / S ratio. The C base indicates a fertile tomato, while the T base indicates a male-sterile tomato. Specific sequence information is as follows:

[0061] The CDS sequence of ABCG (Solyc02g081870) in the wild currant tomato LA1589 material with normal tomato fertility is shown in SEQ ID NO.49.

[0062] SEQ ID NO.49:

[0063] The amino acid sequence of ABCG (Solyc02g081870) in the wild currant tomato LA1589 material with normal tomato fertility is shown in SEQ ID NO.50.

[0064] SEQ ID NO.50:

[0065] The CDS sequence of the ABCG(Solyc02g081870) gene in the tomato male sterility mutant ps-like is shown in SEQ ID NO.51.

[0066] SEQ ID NO.51:

[0067] The amino acid sequence of the ABCG(Solyc02g081870) gene in the tomato male sterility mutant ps-like is shown in SEQ ID NO.52.

[0068] SEQ ID NO.52:

[0069] 6. Application of SNP tags.

[0070] This invention utilizes primers SEQ ID NO.41 and SEQ ID NO.42 to randomly select 22 individual plants from the F2 population for DNA extraction. After PCR amplification, first-generation sequencing is performed, and the DNA is compared with a reference genome. The genotypes of each individual plant are then statistically analyzed. The results are shown in […]. Figure 4 See Table 6. The methods for DNA extraction and PCR amplification are the same as above.

[0071] Table 6. SNP primer genotype statistics In Table 6, the maternal ps-like genotype is T / T, the paternal LA1589 genotype is C / C, and the F2 single plant genotype C / C is a homozygous fertile single plant, T / T is a homozygous sterile single plant, and C / T is a heterozygous fertile single plant.

[0072] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0073] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A molecular marker associated with tomato anther dehiscence, characterized in that, The molecular marker is the nucleotide sequence shown in SEQ ID NO.

51.

2. The application of the molecular marker as described in claim 1, characterized in that, The molecular marker is used in any of the following: 1) Screening or assisted screening of male-sterile tomato mutants; 2) To identify or assist in the identification of male-sterile tomato mutants; 3) Tomato hybridization seed production.

3. The application as described in claim 2, characterized in that, The method for identifying male-sterile tomato mutants is as follows: DNA was extracted from the tomatoes to be identified; Using DNA as a template, PCR amplification was performed using the primer sets shown in SEQ ID NO.41 and SEQ ID NO.42, followed by electrophoresis to obtain the PCR product; If the PCR product is sequenced and the genotype is T / T, then the tomato to be identified is male-sterile.

4. The application as described in claim 3, characterized in that, The DNA of the tomato to be identified was derived from tomato leaves.

5. The application as described in claim 3, characterized in that, The PCR amplification system is as follows: 2×M5 HiPer plus Taq HiFi PCR mix 5μL, DNA 1μL, forward primer 0.5μL, reverse primer 0.5μL, ddH2O 3μL.

6. The application as described in claim 3, characterized in that, The PCR amplification procedure is as follows: Pre-denaturation: 95℃ for 3 min, 1 cycle; Denaturation: 94℃ for 25s, annealing at 55℃ for 25s, extension at 72℃ for 15s / 1kb, 35 cycles; Final extension: 72℃ for 5 minutes, 1 cycle.

7. The application as described in claim 3, characterized in that, After electrophoresis, the fertility of the tomato to be tested can be determined based on the band pattern, as follows: If the band size of the tomato to be identified is consistent with that of the ps-like band, then the tomato to be identified is male-sterile.

8. A reagent for detecting the fertility of tomatoes, characterized in that, The reagent comprises the primer set as described in claim 3.

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