Rapid generation of a tomato male sterility system and its feasible application in hybrid seed production

doi:10.21203/rs.3.rs-2816433/v1

Download PDF

Research Article

Rapid generation of a tomato male sterility system and its feasible application in hybrid seed production

https://doi.org/10.21203/rs.3.rs-2816433/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 22 Aug, 2023

Read the published version in Theoretical and Applied Genetics →

You are reading this latest preprint version

Male sterility enables reduced cost and high seed purity during hybrid seed production. However, progress toward its commercial applicationhas been slow in tomato due to the disadvantages of most natural male-sterile mutants. Here, we developed a practical method for efficient tomato hybrid seed production using a male sterile system with visible marker, which was rapidly generated by CRISPR/Cas9-mediated gene editing. Two closely linked genes, TM6 and DFR, which were reported to be candidates of ms15 (male sterile-15) and aw (anthocyanin without) locus respectively, were knocked out simultaneously in two elite tomato inbred lines. Mutagenesis of both genes generated green hypocotyl male-sterile (GHMS) lines, exhibiting male-sterility across different genetic backgrounds and environmental conditions and showing green hypocotyl due to defective anthocyanin accumulation, which serves as a reliable visible marker for selecting male-sterile plants at the seedling stage. We further proposed a strategy for multiplying the GHMS system and verified its high efficiency in stable male sterility propagation. Moreover, elite hybrid seeds were produced using GHMS system for potential side effects evaluation, and no adverse influences were found on seed yield, seed quality as well as important agronomic traits. This study provides a practical approach for the rapid generation and feasible application of male sterility in tomato hybrid breeding.

tomato

male sterility propagation

visible marker

breeding application

A practical approach for the rapid generation and feasible application of green hypocotyl male-sterile (GHMS) tm6 dfr lines in tomato hybrid breeding was established.

Male sterility is an extensively used strategy during hybrid seed production in crops, as it can enable reduced costs and high seed purity (Chen and Liu, 2014; Kim and Zhang 2018). Tomato (Solanum lycopersicum) is one of the most widely cultivated and commonly consumed vegetables all over the world. Although many recessive genic male-sterile mutants have been reported in tomato (Cheng et al., 2022; Wang et al., 2022), commercial application of male sterility in tomato hybrid breeding is still limited. Many tomato sterility mutants have been discovered and are divided into three categories: functional sterility, structural sterility and sporogenous sterility, as represented by stamenless (sl) (Gomez et al., 1999), positional sterile-2 (ps-2) (Atanassova, 1999), and male sterile-10 (ms10) (Jeong et al., 2014), respectively. To date, several male sterility genes have been cloned. The functional sterility mutant ps-2 conferred nondehiscent anthers due to a single-nucleotide polymorphism (SNP) in the polygalacturonate gene PS-2 (Gorguet, 2009). ms10 was characterized by failed pollen production and an exerted stigma owing to a deletion mutation in a basic-helix-loop-helix (bHLH) transcription factor (Jeong et al., 2014). Another spontaneous sterility mutant, ms32, presented completely impaired pollen and tapetum development and was also found to be controlled by a putative bHLH transcription factor (Liu et al., 2019). Tomato male sterile-15 (ms15) bears flowers with deformed anthers and exerted stigmas, for which the B-class MADS-box gene TM6 was identified as a candidate (Cao et al., 2019). Mutation in another B-class MADS-box gene, GLO2, resulted in male sterility 7B-1 (Pucci et al., 2017).

In contrast to the many theoretical studies on male sterile genes, rare successful application has been reported due to disadvantages of most natural male-sterile mutants. Apart from unstable sterility or unwanted side effects, there are two limitations in common. One limitation lies in the difficulty of male-sterile seed propagation. All of the discovered male sterility traits are controlled by recessive genes, which are usually spread through cross-pollinating homozygous male-sterile plants with heterozygous male-fertile plants. Thus, progeny from the cross is indeed a mixture of 50% male-sterile and 50% male-fertile plants which cannot be distinguished from each other without costly genotyping before transplanting to the field (Atanassova, 2004). Accordingly, the usage of linked early-development-stage morphological markers for male sterility has been proposed to address this problem. This was effectively demonstrated by linkage of ms10 and ms15 with aa (anthocyanin absent) and aw (anthocyanin without), which are both responsible for the impaired anthocyanin production and led to green hypocotyl of seedlings (Clayberg, 1965; Mutschler et al., 1987; Zhang et al., 2016). The other limitation is the time-consuming and laborious efforts for transferring recessive male sterility into elite lines. To complete the whole directional transfer process, at least five generations of backcrossing are needed. Moreover, some other challenging problems, such as false selection and linkage drag, seem to be irradicable.

As a powerful genome editing technology (Aglawe et al., 2018; Gao, 2021), the CRISPR/Cas9 system provides a promising tool to overcome the above limitations to efficiently create male sterility. In this study, by engineering two closely linked genes TM6 and DFR (Cao et al., 2019; Goldsbrough et al., 2004), we rapidly generated a tomato male-sterile system with male sterility and a visible marker (green hypocotyls) which was allowed to identify male sterility at the seedling stage. We further proposed a strategy for propagating the “green hypocotyl” male-sterile plants and verified its stability and efficiency in practice.

Plant materials and growth conditions

All of the tomato inbred lines used were originally developed by our lab. Two inbred lines, TB0993 and TB0249, were subjected to CRISPR/Cas9-mediated gene editing targeting TM6 and DFR, respectively, and gave rise to tm6 dfr double mutants in the T0 generation. Crossing tm6 dfr mutants with corresponding WT plants to obtain T1 and further self-pollination of the T1 plants allowed the generation of T2 offspring. In the T2 population, genotyping for foreign fragments and TM6 as well as the DFR genes was performed to identify transgene-free homozygous tm6 dfr lines (i.e., GHMS/TB0993 and GHMS/TB0249).

Three F2 populations, SP1, SP2 and SP3, were generated following crosses GHMS/TB0993 × TB0993 (WT), GHMS/TB0249 × TB0249 (WT) and GHMS/TB0993 × TB0748 (pink-cherry fruited inbred line). For male sterility stability assessment, the three populations were grown in Tongzhou farm in Beijing (March to May, 2021) and Sanya farm in Hainan province (October to December, 2021), where temperature and relative humidity were automatically measured using recorders at an interval of 30 minutes. During the flowering stage, plant self-pollination was assisted via a “shaking plants” method. For color marker reliability assessment, seedlings of the three F2 populations at the one-leaf stage were cultured in optimal culture conditions (26°C, 150 microeinsteins m^–2 s^–1, 16 L/8 D) or extreme culture conditions (32°C, 50 microeinsteins m^–2 s^–1, 8 L/16 D) in an artificial climate chamber until the fifth leaf developed.

Two BC1F1 populations, BC1F1/TB0993 and BC1F1/TB0249, were generated by crossing a homozygous GHMS line (green, mmaa) with a hemizygous maintainer line (purple, MmAa). The plants were subjected to linkage analysis of the “green hypocotyl” and male sterility together with the above three F2 populations (SP1, SP2 and SP3) through individual genotyping of TM6 and DFR genes.

The GHMS line (green, mmaa) was maintained through a backcross with heterozygous plants (purple, MmAa). To assess the stability and efficiency of the GHMS propagation system, GHMS lines were propagated at Tongzhou farm in Beijing (March 2020 to August 2021) and Sanya farm in Hainan province (September 2019 to February 2021) for four successive generations. During every round of crossing, green and purple plants were visually sorted out at the seedling stage, and recombinants (fertile green plants) were removed based on fruit-set after spontaneous self-pollination on first flower cluster or one retained lateral branch. The first generation (G1) and the fourth generation (G4) were subjected to analysis in terms of genotype composition, seed yield, and seed germination.

Two GHMS-derived F1 hybrids JF101-S and JF501-S were generated following crosses GHMS/TB0993 × TB0994 and GHMS/TB0249 × TB0244 to assess the side effects of GHMS lines on hybrid production. Moreover, the WT-derived F1 hybrids JF101 and JF501 were also generated following crosses TB0993 × TB0994 and TB0249 × TB0244 and used as the controls. For each cross, 200 female plants were used. Evaluation of side effects on agronomic traits of GHMS was performed in tunnel greenhouses at Tongzhou farm (March to August, 2021) and Sanya farm in Hainan province (September 2021 to February 2022). Female parents for cross and hybrids for side effects evaluation were decapitated when the sixth inflorescence developed, and flower and fruit thinning was performed with 4–5 fruits per inflorescence.

CRISPR/Cas9-mediated mutagenesis

The knockout vector was constructed using the binary vector PTX041 as described previously (Deng et al., 2018). Two synthesized sgRNAs targeting the first exon of TM6 (Solyc02g084630) and DFR (Solyc02g085020) were cloned into the PTX041 vector and then introduced into Agrobacterium tumefaciens EHA105 for subsequent transformation into tomato inbred lines TB0993 and TB0249, respectively. PCR was performed for generated T0 plants using primers designed based on the flanking sequence of on-targets as well as predicted off-targets, and the amplicon(s) from each line was then cloned into a high-copy vector for sequencing (Tianyi Huiyuan Bioscience & Technology, Inc. (Beijing, China)).

The primers used are listed in Table S1.

Gene expression analysis

Gene expression analysis was conducted in GHMS/TB0993 and GHMS/TB0993 via quantitative real-time polymerase chain reaction (qRT-PCR) using the corresponding WT plants as controls. Transcripts were examined in hypocotyls and cotyledons at the seedling stage for DFR and in flower buds at the anthesis stage for TM6. Gene expression levels were normalized against those of tomato ACTIN2 calculated using the ΔΔCt method. All experiments were performed with four biological replicates.

The primers used are listed in Table S1.

Pollen vitality analysis

Pollen vitality was determined using the FDA staining method (Li et al., 2011) combined with the germination in vitro method (Song and Tachibana, 1999). For the FDA staining method, fresh stamens taken from flowers at the anthesis stage were squashed to release pollen into 20 µL FDA solution (2 µg ml^− 1) on a glass slide. A Zeiss AX10 fluorescence microscope was used for microscopic observations under fluorescent light conditions. For the germination in vitro method, fresh pollen harvested at the anthesis stage was spread on germination medium consisting of 1% agar, 13% sucrose and 15 mg/l boric acid and then observed using a Zeiss AX10 microscope in a bright field. All experiments were performed with three biological replicates for CRISPR/Cas9-derived mutants (tm6 dfr/TB0993 and tm6 dfr/TB0249) and with thirty biological replicates for three alternative genotyes of TM6 from SP1, SP2 and SP3 populations.

Anthocyanin content determination

The anthocyanin content was determined as described previously (Sun et al., 2020). Approximately 100 mg fresh weight (FW) of collected hypocotyl or young leaf tissues was ground to powder in liquid nitrogen and then put in extraction buffer (1% HCl, 18% 1-propanol and 81% water) overnight incubation at 4°C. The absorbance values of the collected supernatant after centrifugation were determined at wavelengths A535 and A650, and the anthocyanin content was calculated as (A535-A650)/mg FW. All experiments were performed with three biological replicates.

Linkage analysis

Individuals from two BC1F1 populations (BC1F1/TB0993 and BC1F1/TB0249) were genotyped for TM6 and DFR genes, and the recombination fraction was calculated as the percentage of recombinants in all of the plants. Before genotyping, green plants were visually selected, and the selection efficiency of morphological markers for male sterility was expressed as the percentage of mm seedlings in all of the seedlings.

Assessment of side effects on seed yield, seed quality and agronomic traits

At the flowering stage, flowers at anthesis in the second inflorescences were tagged.

Seeds were collected from fruits at the red stage and were subjected to yield and quality analysis. Seed yield was calculated as the average seed number per fruit, which was determined by collecting seeds from 10 fruits of the second inflorescence with 5 replicates. Seed weight was calculated as the average weight per 1000 seeds represented by 5 replicates. Seed germination was determined after 3 days of cultivation in an artificial climate chamber at 30°C. For each assay, 200 seeds were used with three replicates. Seed purity was determined using a male parent-specific molecular marker Ty1 (Table S1) for four F1 hybrids (JF101, JF101-S, JF501 and JF501-S) or using the color marker (green hypocotyls) for JF101-S and JF501-S. For seed germination and seed purity assay, 384 seeds were used and three biological replicates were performed.

Hybrid fruits were collected at the breaker stage and weighed. Their longitudinal diameters (LD) and transverse diameters (TD) were determined using a Vernier caliper, and fruit shape index was calculated as the ratio of TD to LD. At the B + 7 stage (7 days after the breaker stage), fruits were harvested to measure fruit firmness, soluble solid content (SSC), lycopene content and β-carotene content. Flesh firmness was determined using a hardness tester HPE II Fff (Bareiss, Germany), with the results recorded in N. Then, each fruit was cut in half. One half was used to determine the SSC of the juice using a refractometer, with the results recorded as °Brix, and the other half was subjected to lycopene and β-carotene content measurements by HPLC with the method described previously (Zhou et al., 2022). All experiments above were performed with fifteen biological replicates.

Statistical analyses

All data represent the mean value ± standard deviation (SD) of biological replicates. Statistical significance was determined using Student’s t test at the 0.05 (*) level.

Rapid generation of green hypocotyl male sterile lines by the CRISPR/Cas9 system

Most tomato inbred lines develop purple hypocotyls due to anthocyanin accumulation, thus green hypocotyl conferred by aw could serve as an efficient seedling morphological marker for identifying male sterility plants of ms15, which is tightly linked to the aw. TM6 and DFR (Dihydroflavonol 4-reductase) were reported to be the underlying genes for the ms15 and aw locus respectively (Cao et al., 2019; Goldsbrough et al., 2004), although direct genetic evidence are absent. We therefore proposed to employ a modified CRISPR/Cas9 gene-editing system to edit TM6 and DFR simultaneously in two elite inbred lines to generate “green hypocotyl” male sterile lines and to evaluate their usage in our breeding program.

For this purpose, two synthetic gRNAs targeting the first exon of each gene were designed and cloned into the pTX041 vector (Fig. 1), as described in our previous study (Deng et al., 2018). The resulting construct was transformed into one pink-fruited tomato inbred line TB0993 and one red-fruited tomato inbred line TB0249 through Agrobacterium-mediated transformation.

After the recovery of transgenic plants, 10 T0 plants for TB0993 and 13 T0 plants for TB0249 were analyzed for mutations detection within the target regions (Fig. 1B-E). Genome-editing event analysis by sequencing indicated that seven TB0993 transgenic plants carried homozygous or biallelic double mutations with small indels (≤ 12 bp) at both target sites. Likewise, among 13 TB0249 T0 plants, six plants carried homozygous or biallelic double mutations. Moreover, we sequenced the potential off-target sites predicted by Cas-OF Finder (Bae et al., 2014) in all identified mutated T0 plants, and no off-target mutations were found at these sites. These results highlight the high editing efficiency and specificity of the CRISPR/Cas9 system used in this study.

After removing the transgene insertions by back-crossing, we selected two homozygous double-mutated lines tm6 dfr/TB0993 #1 and tm6 dfr/TB0249 #1 for phenotypic analysis. Mutations in TM6 and DFR in these lines were predicted to introduce premature stop codons and cause undetectable expression of each gene probably owing to nonsense-mediated mRNA decay (Fig. S1). There was no difference in growth and development between wild-type (WT) and tm6 dfr plants until the flowering stage. At flowering, tm6 dfr lines were found to produce obviously twisted stamens and failed to set fruit (Fig. 2A, D and Fig. S2A, D). The exhibiting male sterility was further supported by the fluorescein diacetate (FDA) staining assay and pollen germination assay, which did not detect viable pollen in the tm6 dfr flowers (Fig. 2B, C and Fig. S2B, C). In contrast, tm6 dfr plants produce normal pistils, and they set comparable fruits and seeds when pollinated with WT pollen (Fig. 2E, F and Fig. S2E, F). These results demonstrated that the tm6 dfr plants are fully male sterile and have application prospects for hybrid seed production in tomato.

Although tm6 dfr plants did not have any aberrant phenotypes during seedling growth and development as expected, we observed obvious defects in anthocyanin accumulation in the tm6 dfr plants when compared with the WT plants (Fig. 2G, H and Fig. S2G, H). The differences in anthocyanin accumulation, as represented by seedling color (i.e., purple in WT versus green in the mutants), could be seen as early as 2–3 days after germination. The “green hypocotyls” in tm6 dfr plants were stable during the whole seedling stage. In addition, various color differences between the WT and tm6 dfr plants were also observed in both vegetative and floral tissues throughout the entire life cycle (Fig. 2I and Fig. S2I). These results confirmed previous findings that DFR functions in anthocyanin accumulation in tomato and indicated that the “green hypocotyls” in the tm6 dfr plants can serve as a visible marker. We therefore renamed the tm6 dfr plants GHMS (green hypocotyl male sterile) for further analysis.

Phenotypic stability of GHMS across genetic backgrounds and environmental conditions

To evaluate the application potential of the GHMS lines in tomato hybrid breeding, we sought to examine the phenotypic stability across various genetic backgrounds and environmental conditions. Three GHMS-derived F2 segregating populations were generated: (i) SP1, derived from a cross between GHMS/TB0993 and its parental line TB0993, (ii) SP2, derived from a cross between GHMS/TB0249 and its parental line TB249, and (iii) SP3, derived from a cross between GHMS/TB0993 and a pink cherry-fruited inbred line TB0748.

The three populations were grown for male sterility assessment on Tongzhou farm in Beijing (March to May, 2021) and Sanya farm in Hainan province (October to December, 2021), which represented contrasting environmental conditions, as shown in Fig. 3A. The male fertility of flowers from the first three inflorescences was examined in terms of pollen vitality and fruit set. Under both growth conditions, all homozygous tm6 (mm) plants from the three F2 populations, as genotyped by mutation-specific molecular markers, displayed male sterility; they failed to produce viable pollen grains and set seeded fruits, in contrast to normally developed pollens and seeded fruits of heterozygous tm6 (Mm) or homozygous Tm6 (MM) plants (Fig. 3A). These results demonstrated that the male sterility of GHMS is stable across genetic backgrounds and environmental conditions.

Anthocyanin production was reported to be affected by genetic, environmental, and nutritional cues (Cominelli et al., 2008; Butelli et al., 2012; Das et al., 2012; Hodges and Nozzolillo, 1995). Thus, we carefully analyzed the correlation between the dfr mutations and anthocyanin accumulation (seedling color) in the three F2 populations under different growth conditions (Fig. 3B and Fig. S3). When grown under optimal conditions (26°C, 150 microincisions m^–2 s^–1, 16 L/8 D), all homozygous dfr (aa) seedlings identified from the three populations displayed anthocyanin-deficient green color, with conspicuous color differences from the heterozygous dfr (Aa) seedlings. Although a wide range of variation in anthocyanin accumulation was observed in Aa individuals, apparent discrepancies between purple and green plants allow them to be easily distinguished as aa plants (Fig. S4A). These results indicate that, under optimal conditions, seedling color can serve as a reliable marker to select aa plants. To explore environmental effects, F2 seedlings were subjected to extreme conditions with weak light and high temperature (32°C, 50 microincisions m^–2 s^–1, 8 L/16 D). Under this growth condition, anthocyanin accumulation was obviously reduced in Aa plants, which attenuated color differences between aa and Aa plants and led to an ~ 10%-15% decrease of the efficiency for selecting aa plants (Fig. S4B). This result implied that adverse conditions should be avoided during the seedling stage in practice to maintain highly effective screening of aa plants by color markers.

Linkage analysis of the “green hypocotyl” and male sterility

We next discussed the use of the GHMS line in tomato hybrid breeding. The classic approach for the use of recessive genic male sterility is to cross-pollinate homozygous male-sterile plants with heterozygous male-fertile plants. Progeny from the cross is a mixture of segregating male sterile (50%) and fertile (50%) plants. The use of linked genetic markers expressed at the seedling stage, such as the “green hypocotyls” of our GHMS line, allows for the visual identification of male-sterile plants from the mixture population before transplanting to the field. The selection efficiency for male sterility by “green hypocotyls” largely depends on the recombination frequency between the TM6 and DFR genes. Thus, we used two GHMS-derived BC1F1 populations, BC1F1-TB0993 and BC1F1-TB0249 (Table 1), to perform the linkage analysis of TM6 and DFR. A total of 8 recombinants were detected among 384 BC1F1-TB0993 individuals, while 7 recombinants were detected among 384 BC1F1-TB0249 individuals. The recombination fractions in the two populations were 2.08% and 1.82%, respectively. Accordingly, the selection efficiency for male sterility by “green hypocotyls” in the two populations was calculated to be approximately 97.50% and 97.87%, respectively. These analyses suggested that the “green hypocotyl” of our GHMSprovides a reliable visible marker for the selection of male sterility.

Table 1

Recombination frequency between the *TM6* and *DFR* based on two BC1F1 populations
	aa		Aa		Recombination fraction	Screening efficiency
	msms	Msms	msms	Msms	Recombination fraction	Screening efficiency
BC1F1-TB0993	195	5	3	181	2.08	97.50
BC1F1-TB0249	184	4	3	193	1.82	97.87
Note: Selection efficiency was expressed as the percentage of mm plants in all the visually selected green (aa) seedlings

Propagation strategy of the GHMS seeds

We next proposed a strategy to propagate and identify GHMS plants for hybrid seed production without costly genotyping (Fig. 4A). Homozygous GHMS plants (green, mmaa) were cross-pollinated with WT pollen to generate a hemizygous maintainer line (purple, MmAa). The cross between mmaa and MmAa produced the generation 1 (G1) progeny population (hereafter named “the propagation population”), which was predicted to segregate half green (consisting of 98% mmaa and 2% Mmaa) and half purple plants (consisting of 98% MmAa and 2% mmAa). The two groups of plants could be easily selected by seedling color and were grown separately. At the flowering stage, the first flower clusters of the green plants were allowed to be self-pollinated, and the male fertile plants with self-pollinating fruits (the 2% Mmaa) were removed. The remaining green plants (98% mmaa) were then cross-pollinated with pollen from the purple plants (please note that only the MmAa purple plants can produce viable pollen), which gave rise to the new generation of the propagation population. Theoretically, the above-mentioned procedures can be operated iteratively, and the genetic composition of the propagation population should be similar over generations. To assess the stability and efficiency of this propagation strategy, we performed the propagation procedure for 4 successive generations and genotyped the individuals in G1 and G4. As expected, the composition of genotypes in G4 resembled that in G1 (Fig. 4B). This result indicated that the GHMS seeds can be stably and effectively propagated using our proposed strategy, and this strategy can be used to produce hybrid seeds at a commercial scale.

Side effects analysis of using GHMS lines in hybrid production

To further evaluate the application of our GHMS lines in hybrid tomato breeding, we used the GHMS lines (GHMS/TB0993 and GHMS/TB0249) to produce hybrid seeds of two elite F1 varieties, Jingfan101 (JF101, TB0993 × TB0994) and Jingfan501 (JF501, TB0249 × TB0244), which were developed by our group. The GHMS/TB0993 and GHMS/TB0249 lines were propagated, and seedlings with “green hypocotyls” were selected for subsequent hybrid seed production. At the flowering stage, the male fertile plants with self-pollinating fruits were removed. The remaining plants were then cross-pollinated with the male parent inbred lines (i.e., TB0994 for JF101 and TB0244 for JF501) to generate the F1 hybrid seeds. To compare the performances with those of the original hybrids, the GHMS-derived F1 hybrids were designated JF101-S and JF501-S, respectively.

Seed yield and quality are critical for hybrid seeds. As shown in Fig. 5 and Fig. S5, the seed yield, weight and germination rate were comparable in the WT- and GHMS-derived F1 hybrids. However, as expected, the GHMS-derived F1 hybrids had obviously higher seed purity than the WT-derived F1 hybrids (Fig. 5A and Fig. S5A). Other important agronomic traits were also tested (Fig. 5B, C and Fig. S5B, C). The fruit ripening time of JF101-S and JF501-S was similar to that of WT-derived F1 hybrids. In addition, no significant difference was observed in single fruit weight, yield per plant, as well as fruit shape. Fruit quality was further examined. The main quality indices of ripe fruits, including fruit firmness, levels of lycopene, β-carotene and total soluble solids content (°Brix), did not show any significant difference between the GHMS- and WT-derived F1 hybrids. These results suggest that GHMS-produced hybrids do not have any negative effects on variety performance and can meet the quality requirements of commercial tomato production. Thus, the GHMS system can be well applied to the commercial production of hybrid seeds.

As one of the most consumed vegetables all over the world, tomato provides a classical model system for studying crop domestication, molecular breeding, and fruit biology (Du et al., 2017; Xia et al., 2021). Most commercial varieties of tomato are F1 hybrids due to their superior over parents in terms of yield, disease resistance and environmental fitness (Perez-Prat and van Lookeren Campagne, 2002). During the process of hybrid seed production, artificial emasculation results in increased labor expenses. The utilizing male sterility to produce hybrids is an efficient approach to reduce the cost and ensure high varietal purity (Chen and Liu, 2014; Kim and Zhang 2018; Perez-Prat and van Lookeren Campagne, 2002). Many spontaneous male-sterile mutants have been reported in tomato (Wang et al., 2022; Jeong et al., 2014; Liu et al., 2019; Cao et al., 2019; Pucci et al., 2017). However, some inherent disadvantages prevent their application in hybrid seed production. Apart from unstable sterility or unwanted side effects, there are two limitations in common.

One limitation is the time-consuming and laborious efforts for transferring recessive male sterility into an elite inbred line. To complete the whole directional transfer process, at least five generations of backcrossing are needed. Moreover, some other challenging problems, such as false selection and linkage drag, seem to be irradicable. CRISPR/Cas9 genome editing technology has provided alternative opportunities for breeders to introduce male sterility into elite varieties within one generation at low costs and with no linkage drag. For example, we previously generated novel male-sterile tomato lines by CRISPR/Cas9-mediated mutagenesis of the stamen-specific gene SlSTR1 in several elite inbred backgrounds (Du et al., 2020). Similarly, rapid generation of male sterility in elite inbred lines was also achieved in this study by knocking out the TM6 which was reported to be a candidate gene for ms15 (Cao et al., 2019). The tm6 lines generated in the present study developed deformed stamens, with no viable pollen observed at the anthesis stage. These phenotypes mimic ms15, indicating that TM6 indeed underlies ms15 locus. Moreover, we found that the male sterility caused by the tm6 mutation was very stable under various genetic backgrounds and environmental conditions, highlighting its valuable and extensive application potential in tomato hybrid seed production.

The other limitation for applying genic male sterility to hybrid seed production lies in the difficulty of propagating pure male-sterile seeds at a large scale. Usually, homozygous male-sterile (ms/ms) seeds are propagated by cross-pollinating ms/ms plants with heterozygous male-fertile (Ms/ms) plants. The resulting F1 progeny is mixture of 50% ms/ms plants and 50% Ms/ms plants. To produce F1 hybrid seeds, all of the Ms/ms plants must be removed based on fertility identification during flowering stage, which is labor-intensive and resource-consuming. Two strategies have been proposed to overcome this difficulty. The first strategy is to create transgenic maintainers by transforming the fertility restoration gene linked with a pollen-lethality gene or a seed-color gene into the male-sterile plant (Perez-Prat and van Lookeren Campagne, 2002). When cross-pollinating the male-sterile line (ms/ms, −/−) with hemizygous maintainer (ms/ms, Maintainer/−) pollens, it would generate only the male-sterile seeds (in the case of pollen-lethality maintainer) or 50% male-sterile seeds and 50% color maintainer seeds which can be visually sorted out according to seed color differences. Using such kinds of transgenic maintainers to multiply male sterility have been recently reported in maize and rice (Kim and Zhang 2018; Chang et al., 2016; Wu et al., 2016; Zhang et al., 2018; Wan et al., 2019; Cai et al., 2022). Using a similar strategy, we recently developed a transgenic maintainer with a seedling color marker to propagate our CRISPR/Cas9-generated tomato male sterile line (Du et al., 2020). The second strategy to obtain a large quantity of recessive genic male sterile plants is the use of closely linked morphological markers expressed in seedling that can help remove fertile plants before transplantation. This approach was effectively demonstrated by linking ms10 with aa or linking ms15 with aw in tomato (Clayberg, 1965; Mutschler et al., 1987; Zhang et al., 2016). In this study, the candidate gene DFR for aw locus (Goldsbrough et al., 2004) was knocked out using CRISPR/Cas9 system and it led to no anthocyanin accumulation in hypocotyls (green hypocotyls), which serves as a visible marker for selecting male-sterile plants of tm6 at the seedling stage at high accuracy (~ 98%). On the other hand, this provides genetic evidence to support the idea that DFR underlies aw locus. We must note that, using a similar approach, a recent study generated ms10 male sterile lines with two different seedling markers (Liu et al., 2021). However, in that study, the authors did not provide convincing data in terms of the propagation method and application of these sterile males in hybrid seed production. In contrast, we proposed a strategy to propagate green hypocotyl male sterile seeds without costly genotyping and verified its feasible application. Our data demonstrated that the proposed propagation method is stable and efficient, and the developed GHMS lines are suitable for tomato hybrid seed production. Especially, it had no visible negative influence on key agronomic traits of elite hybrids. However, although the “green hypocotyls” can help select the male-sterile plants at the seedling stage, the selection efficiency of “male sterility” by the “green hypocotyl” in our GHMS system was not 100% due to the incomplete linkage between the TM6 and DFR (Table 1). Further searches for co-segregated genetic markers of male sterility are needed to improve this system.

Collectively, we developed a green hypocotyl male-sterile system by using the advantages of powerful CRISPR/Cas9 technology. More importantly, we further present a practical proposal for feasible application of this system to tomato hybrid production. With stable male sterility across different genetic backgrounds and environmental conditions and no adverse side effects on seed quality and agronomic traits, widespread applications of this system can be anticipated. The practical study of our work could be extended to other vegetable crops for rapid male sterility creation and its feasible application in hybrid seed production.

Data availability

The data generated are available in the article and the supplementary materials.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2022YFD1200502), Beijing Municipal Science and Technology Project (Z211100004621001), Beijing Joint Research Program for Germplasm Innovation and New Variety Breeding (G20220628003) and Key-Area Research and Development Program of Guangdong Province (2018B020202006).

Funding

National Key Research and Development Program of China (2022YFD1200502), Beijing Municipal Science and Technology Project (Z211100004621001), Beijing Joint Research Program for Germplasm Innovation and New Variety Breeding (G20220628003) and Key-Area Research and Development Program of Guangdong Province (2018B020202006).

Author contributions

CB. L. and CY. L. conceived and supervised the project. M. Z. performed the experiments. W. Z. helped perform the experiments. L. D., MM. D., GL. Y., MY. M and CL. S. analyzed the data. M. Z. and MM. D. wrote the manuscript. All authors read and approved the manuscript.

Conflict of interest

The authors declare that they have no conflicts of interest.

Aglawe SB, Barbadikar KM, Mangrauthia SK, Madhav MS (2018) New breeding technique “genome editing” for crop improvement: applications, potentials and challenges. Biotech:8, 336
Atanassova B (1999) Functional male sterility ps-2 in tomato (Lycopesicon esculentum Mill.) and its application in breeding and hybrid seed production. Euphytica:107, 13–21
Atanassova B (2004) Genic male sterility and its application in tomato (Lycopersicon esculentum mill.) hybrid breeding and hybrid seed production. In: Proceedings of the IIIrd balkan symposium on vegetable and potatoes:729, 45–51
Bae S, Park J, Kim JS (2014) Cas-OF Finder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics:30, 1473–1475
Butelli E, Licciardello C, Zhang Y, Liu J, Mackay S, Bailey P, Reforgiatorecupero G, Martin C (2012) Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. Plant Cell:24, 1242–1255
Cai DR, Zhang ZG, Zhao L, Liu J, Chen H (2022) A novel hybrid seed production technology based on a unilateral cross-incompatibility gene in maize. Sci. China Life Sci:3, 595–601
Cao X, Liu XY, Wang XT, Giang TV, Wang J, Liu XL, Sun S, Wei K, Wang XX, Gao JC, Du YC, Qin Y, Guo YM, Huang ZJ (2019) B-class MADS-box TM6 is a candidate gene for tomato male sterile-15²⁶. Theor Appl Genet:132, 2125–2135
Chang ZY, Chen ZF, Wang N, Deng XW (2016) Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene. Proc Natl Acad Sci:113, 14145–14150
Chen L, Liu YG (2014) Male sterility and fertility restoration in crops. Annu. Rev. Plant Biol:65, 1473–1475
Cheng ZH, Song WY. Zhang XL (2022) Genic male and female sterility in vegetable crops. Hortic Res:uhac232
Clayberg CD (1965) A linked seedling marker for ms15. Rep Tomato Genet Crop:15,29–30
Cominelli E, Gusmaroli G, Allegra D, Galbiati M, Wade HK, Jenkins GI, Tonelli C (2008) Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana. J. Plant Physiol:165, 886–894.
Das PK, Shin DH, Choi SB, Park YI (2012) Sugar-hormone cross-talk in anthocyanin biosynthesis. Mol cells:34, 501–507
Deng L, Wang H, Sun CL, Li Q, Jiang HL, Du MM, Li CB, Li CY (2018) Efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system. J Genet Genomics:45, 51–54
Du MM, Zhou M, Deng L, Li CY, Li CB (2017) Current status and prospects on tomato molecular breeding — from gene cloning to cultivar improvement. Acta Horticulturae Sinica:44, 581–600
Du MM, Zhou K, Liu YY, Deng L, Zhang XY, Lin LH, Zhou M, Zhao W, Wen CL, Xing JY, Li CB, Li CY (2020) A biotechnology‐based male‐sterility system for hybrid seed production in tomato. Plant J:102, 1090–1100
Gao, CX (2021) Genome engineering for crop improvement and future agriculture. Cell:184, 1621–1635
Goldsbrough A, Belzile F, Yoder J I (1994) Complementation of the tomato anthocyanin without (aw) mutant using the dihydroflavonol 4-reductase gene. Plant Physiol:105, 491–496
Gomez P, Jamilena M, Capel J, Zurita S, Angosto T, Lozano R (1999) Stamenless, a tomato mutant with homeotic conversions in petals and stamens. Planta:209, 172–179
Gorguet B, Schipper D, van Lammeren A., Visser RGF, van Heusden AW (2009) ps-2, the gene responsible for functional sterility in tomato, due to non-dehiscent anthers, is the result of a mutation in a novel polygalacturonase gene. Theor Appl Genet:118, 1199–1209
Hendelman A, Kravchik M, Stav R, Frank W, Arazi T (2016) Tomato HAIRY MERISTEM genes are involved in meristem maintenance and compound leaf morphogenesis. J Exp Bot:67, 6187–6200
Hodges DM, Nozzolillo C (1995) Anthocyanin and anthocyanoplast content of cruciferous seedlings subjected to mineral nutrient deficiencies. J. Plant Physiol:147, 749–754
Jeong H.J, Kang JH, Zhao M, Kwon JK, Choi HS, Bae JH, Lee H, Joung YH, Choi D, Kang BC (2014) Tomato male sterile 10³⁵ is essential for pollen development and meiosis in anthers. J Exp Bot:65, 6693–6709
Kim YJ, Zhang D (2018) Molecular Control of male fertility for crop hybrid breeding. Trends Plant Sci: 23, 53–65
Li X (2011) Pollen fertility/viability assay using FDA staining. Bio Protoc. 1, e75
Liu JW, Wang SF, Wang H, Luo B, Cai YY, Li XD, Zhang YF, Wang XF (2021) Rapid generation of tomato male-sterile lines with a marker use for hybrid seed production by CRISPR/Cas9 system. Mol Breed:41, 25
Liu XY, Yang MX, Liu XL, Wei K, Cao X, Wang XT, Wang XX, Guo YM, Du YC, Li JM, Liu L, Shu JS, Qin Y, Huang ZJ (2019) A putative bHLH transcription factor is a candidate gene for male sterile 32, a locus affecting pollen and tapetum development in tomato. Hortic. Res:6, 88
Mutschler M, Tanksley S, Rick CM (1987) Linkage maps of the tomato (Lycopersicon esculentum). Rep Tomato Genet Crop:37,5–34
Perez-Prat E, van Lookeren Campagne MM (2002) Hybrid seed production and the challenge of propagating male-sterile plants. Trends Plant Sci:7, 199– 203
Pucci A, Picarella ME, Mazzucato A (2017) Phenotypic, genetic and molecular characterization of 7B-1, a conditional male-sterile mutant in tomato. Theor Appl Genet 130:2361–2374
Song J, Nada K, Tachibana S (1999) Ameliorative effect of polyamines on the high temperature inhibition of in vitro pollen germination in tomato (Lycopersicon esculentum Mill.). Sci Hortic:80, 203–212
Sun CL, Deng L, Du MM, Zhao JH, Chen Q, Huang T, Jia HL, Li CB, Li CY (2020) A transcriptional network promotes anthocyanin biosynthesis in tomato flesh. Mol Plant:13, 42–58
Wan XY, Wu SW, Li ZW, Dong ZY, Li X, Ma AB, Tian YH, Li JP (2019) Maize genic male-sterility genes and their applications in hybrid breeding: progress and perspectives. Mol Plant:12, 321–342
Wang J, Li MZ, Zhuo SB, Liu Y (2022) Mitogen-activated protein kinase 4 is obligatory for late pollen and early fruit development in tomato. Hortic Res:Uhac048
Wu YZ, Fox TW, Trimnell MR, Wang LJ, Xu RJ, Cigan AM, Huffman GA, Garnat CW, Hershey H, Albertsen MC (2016) Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross-pollinating crops. Plant Biotechnol J:14, 1046–1054
Xia XH, Cheng XH, Li R, Yao JN, Li ZG, Cheng YL (2021) Advances in application of genome editing in tomato and recent development of genome editing technolog. Theor Appl Genet:134, 2727–2747
Zhang DF, Wu SW, An XL, Xie K (2018) Construction of a multicontrol sterility system for a maize male-sterile line and hybrid seed production based on the ZmMs7 gene encoding a PHD-finger transcription factor. Plant Biotechnol J:16, 459–471
Zhang LY, Huang ZJ, Wang XX, Gao JC, Guo YM, Du YC, Hu H (2016) Fine mapping and molecular marker development of anthocyanin absent, a seedling morphological marker for the selection of male sterile 10 in tomato. Mol Breed:36, 107
Zhou M, Deng L, Guo SG, Yuan GL, Li CY, Li CB (2022) Alternative transcription and feedback regulation suggest that SlIDI1 is involved in tomato carotenoid synthesis in a complex way. Hortic Res:Uhab04

TableS1andFig.S1S5.pdf

Download PDF

Journal Publication

published 22 Aug, 2023

Read the published version in Theoretical and Applied Genetics →

Reviewers agreed at journal
07 May, 2023
Reviewers invited by journal
05 May, 2023
Editor assigned by journal
17 Apr, 2023
First submitted to journal
16 Apr, 2023

You are reading this latest preprint version

Rapid generation of a tomato male sterility system and its feasible application in hybrid seed production

Status:

Journal Publication

Version 1

Abstract

Figures

Key Message

Introduction

Materials and methods

Results

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1