CHEN Chen , CUI Qing-zhi HUANG San-wen, WANG Shen-hao, LIU Xiao-hong LU Xiang-yang , ,CHEN Hui-ming , TIAN Yun ,

1 College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, P.R.China

2 Hunan Vegetable Research Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, P.R.China

3 Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of the Ministry of Agriculture/Sino-Dutch Joint Laboratory of Horticultural Genomics, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

4 Key Laboratory for Agricultural Biochemistry and Biotransformation of Hunan Provincial University, Changsha 410128, P.R.China

5 Hunan Co-Innovation Center for Utilization of Botanical Functional Ingredients, Changsha 410128, P.R.China

1. Introduction

Cucumber is a cucurbitaceous annual vine. Cucumber is not only an important vegetable crop worldwide, but is also a model for the study of sex expression (Chen et al. 2011).The genetic basis of cucumber is very narrow (Staub et al.1987). Cucumber has a relatively small genome size(350 Mbp), and its genome was sequenced in 2009 (Huang et al. 2009). Only a few genes related to the fruit length and fruit peel color have been identified in cucumber (Zhou et al. 2015; Liu et al. 2016; Wang et al. 2017). However,available tools and resources for functional genomics studies in cucumber are still very limited. For example, an efficient cucumber genetic transformation system is lacking(Wang et al. 2015). Mutant library construction is one direct and effective way for performing gene functional studies.

Several methods have been employed for plant mutant library construction through physical, chemical or biological(insertional) approaches. Physical mutagenesis uses x-ray,γ-ray or other physical elements requiring specialized machinery that is potentially difficult to operate (Sagan et al.1995). Insertional mutagenesis uses T-DNA, transposon and other insertion elements that are randomly inserted into the genome to produce mutations, requiring a practicable genetic transformation system (Lo et al. 2016). Chemical mutagenesis primarily utilizes sodium azide, ethyl methyl sulfone (EMS) and other chemical reagents, among which the EMS mutagenesis method has the advantages of high mutation rates, less chromosome aberration, simple operation and no requirement for genetic transformation,making it used widely (Greene et al. 2003).

EMS mutagenesis has been widely used in various crops for mutant library development including rice, Arabidopsis thaliana, wheat, tomato, soybean, melon and other crops;the mutants generated can not only serve as an important functional genomics tool, but also have practical use in crop breeding (Menda et al. 2004; Chen et al. 2013; Galpaz et al.2013; Henry et al. 2014; Tsuda et al. 2015; Li et al. 2017).However, reports on the construction and application of cucumber mutant library are very limited. No cucumber mutant library is available for public use. In this study,we conducted EMS mutagenesis in the northern China ecotype cucumber inbred line 406 for the construction of a mutant library. We identified a number of mutants with morphological changes related to plant architecture,leaves, floral organs and fruit. In addition, through genetic analysis, we revealed that the short-fruit and yellow-green fruit peel mutations were both under the control of a single recessive gene.

2. Materials and methods

2.1. Plant materials

The parental line used for EMS mutagenesis was the northern China ecotype cucumber inbred line 406. This monoecious line had good resistance to multiple diseases and high yield. Seeds of inbred line 406 were provided by the Hunan Vegetable Research Institute, Hunan Academy of Agricultural Sciences, China.

2.2. Pre-experiment and optimization of EMS concentrations for mutagenesis

Good quality seeds of inbred line 406 (water content and germination rate were 7 and 100%, respectively) were soaked in distilled water in an erlenmeyer flask at 25°C for 12 h. After pouring off the water, EMS (Sigma M-0880,USA) of different concentrations was added to the container for additional 12 h with continuous stirring with a magnetics stirring bar. Then the treated seeds were thoroughly washed in running water for 4 h and dried on paper towels. In order to evaluate the effect of EMS on the M1and M2generations,the 50% lethal dosage (LD50) was determined. All treated seeds were cultivated in 0.05 mol L-1phosphate buffer solution (pH=7.4) with different EMS concentrations: 0(control), 0.5, 0.8, 1.1, 1.4, 1.7, 2.0, and 3.0%, respectively.And each EMS concentration group had three replications and each replication treated 200 seeds. The treated seeds were placed in a 25°C incubator for germination.Seed germination rate was collected on the day when the germination of the control group reached to 100%.

2.3. Development, morphological characterization,and genetic analysis of EMS mutagenesis population

In 2011 spring, 10 000 cucumber seeds of inbred line 406 were treated with 1.5% EMS for 12 h at 25°C. Germinated seeds were planted in nursery trays with herbaceous pea, and the seedlings (M1) were transplanted into netted greenhouses 45 days later. The M1plants were self-pollinated to create M2families (one or two fruits per plant). In the spring of 2012, we planted 15-20 plants per M2family. Mutant plants were self-pollinated to obtain the mutant seeds (M3).

Morphological observation of M2and M3plants were performed both at seedling (3 weeks after sowing) stage in a nursery tray and at adult plant stage in the greenhouse,respectively. Segregation of wild and mutant plants was counted in each M2family to infer the inheritance mode of the mutation.

To investigate the inheritance mode of mutants, in the spring of 2012, we crossed the short fruit mutant and yellow-green fruit peel mutant with the wild type line 406,respectively, to produce F1generations. Then, in the autumn of 2012, the F1seeds were planted and the F1plants were self-pollinated to generate F2populations, which were used to observe in 2013.

3. Results

3.1. ldentification of optimal EMS concentrations for mutagenesis in inbred line 406

We tested seven EMS concentrations and examined their effects on the germination of cucumber seeds. Expectedly,the germination rate was negatively correlated with the EMS concentration (r=?0.953; P=0.00013) (Appendix A). The germination rate decreased gradually with the increase in EMS concentration, and the LD50concentration for EMS mutagenesis in inbred line 406 was approximately 1.5%EMS. Thus, this concentration was used for large-scale EMS treatment of inbred line 406 seeds to develop the mutant library.

3.2. Observation and analysis of M1 traits

Preliminary experiments indicated that 1.5% EMS was the optimal concentration for mutagenesis. Of the 10 000 cucumber seeds treated with 1.5% EMS, 5 400 germinated,and 4 000 survived after transplantation, so the survival rate of seedling was up to 40%. One or two fruits were obtained per plant following self-pollination, 1 980 fruits were harvested and only 552 fruits contained seeds. So,the pollen fertility of M1generation was 27.9%. Overall,through our observation of M1plants throughout the entire growth period, we found that EMS treatment resulted in significant physiological damages to the plants which could be seen from the significant developmental delay, yellowing and dwarfing plants, leaf and flower deformity, and chimera(Fig. 1).

3.3. Phenotypic variations among M2 mutagenesis population

Among 5 051 plants from 552 M2families, 631 exhibited significant mutant phenotypes as compared with the wild type with a phenotypic mutation rate around 12.49%.Morphological changes were observed in plant architecture,leaves, floral organs, fruits and other horticulturally important traits (Table 1). Plants carrying multiple mutations were also observed.

Plant architecture mutations accounted for about 22.5%of all observed morphological changes. The majority of plant architecture-related mutations were dwarfing. Often dwarf plants were also associated with other morphological changes such as wrinkled leaves, incompletely developed male and female flowers that resulted in male and/or female sterility. Mutant plants varied in branch numbers from none with a short petiole to two lateral branches. Some mutants had flat stems with shortened internodes, and a few exhibited multiple tendrils. Still, some mutant plants had no growth point in the early development stage (Fig. 2).

Among all morphological mutations observed, leaf mutants had the highest proportion which was 33.6%. The leaf mutations included changes in leaf color, size, shape and bitterness. Among the leaf color mutants, the colors varied from light green, to pale yellow, and to dark green.Changes in leaf shape and size included medium- and small-sized leaves; inner-rolled leaves; heart-shaped leaves;wrinkled leaves; triangular leaves; nearly round leaves;sharp leaf tips; large leaves; chrysanthemum leaves; and inner-rolled leaf edges, etc. The wild type line 406 had bitter leaves, and bitter-free leaf mutations were identified in the M2families (Fig. 3).

Fig. 1 Representative mutant phenotypes at M1 generation. A, yellow cotyledon. B, leaf deformity. C, inner-rolled leaf. D, leaf color chimera. E, clustered male flowers. F, dwarf plant.

Table 1 Statistics on M2 population mutation types of the cucumber 406 mutant library

Floral organ mutations accounted for about 17.0%of total observed mutations which included mutations in both flower shape and sex expression. We identified an androecious mutant plant in which no single female flower could be found in the first 40 nodes of the main stem. The flower sepals of some plants turned into small green leaves,and the male flowers on the mutant developed incomplete bisexual flowers with reduced size in the pistil and stigma.These mutants seem female sterile since no seeds could be found in fruits from self-pollination. In petal mutants,the petals increased or decreased their sizes or their colors became light yellow in color. Some mutants manifested male and female flower developmental malformations such as elongated or cracked stigmas, undeveloped or deformed stamens, clustered male flowers and twin female flowers(Fig. 4).

Finally, fruit mutations accounted for 26.9% of observed mutations including fruit peel color, fruit shape and fruit pulp color. Among the fruit mutations, changes in fruit-shape were the most common including slender fruit lengths;shortened fruit with reduced stems; shortened fruit with thickened stem; oval fruit; fruits with reduced warts or no warts; glossy or smooth fruit surfaces. Fruit peel color mutations varied from light yellow, light green, yellow-green to dark green. Yellow flesh color was the only flesh color mutation observed (Fig. 5).

Fig. 2 Phenotypes of representative plant architecture-related mutants in M2 generation. A, wild type plant. B, compact plant.C, long-branched plant. D, flat stem plant. E, extremely dwarf plant. F, plant with no growth points. G, plant with multiple tendrils.H, plant with a whitened main vine. I, plant with shortened internodes.

Based on segregation among M3plants, we found that about 88% mutant traits observed in M2were inheritable.Some examples included yellow leaf, heart-shaped leaf,fruit length and stem thickness mutations. However,we also found that the bisexual flower mutation was not inheritable.

Fig. 3 Phenotypes of leaf mutants of M2 generation. A, yellow-green leaf color (left for the wild type; right for mutant plant). B, light green leaf color. C, heart-shaped leaf. D, chrysanthemum leaf. E, wrinkled leaf. F, bitter-free leaf. G, dark green and wrinkled leaf. H, round leaf. I, sharp leaf tips. J, inner-rolled leaf.

Fig. 4 Phenotypes of typical floral organ mutants in M2 generation. A, wild type. B, all male flowers. C, twin female flowers.D, clustered male flowers and bisexual flowers. E, calyx mutant male flowers (left for the wild type; right for the mutant). F, size of mutant male flowers (the biggest one for the wild type). G, mutant female flowers (left for the wild type; right for the mutant).H, mutant male flowers (left for the wild type; right for the mutant). I, calyx mutant female flowers (left for the wild type; right for the mutant).

3.4. Inheritance short fruit and yellow fruit peel mutations

We crossed the short fruit mutant with wild type line 406.The F1exhibited long fruit that was the same as the wild type. Among 245 F2plants observed, 180 and 65 were born long and short fruits, respectively (Table 2). Similarly, we crossed yellow-green fruit peel mutant with the wild type plant. The F1fruit color was the same as the wild type fruit.In F2, 150 and 48 F2plants had fruits with yellow-green and green fruit peels, respectively (Table 3). In both cases, the segregations were consistent with a single recessive gene underlying the fruit length and fruit skin color mutations,respectively.

4. Discussion

Mutant library is an important tool for functional genomics research. Although EMS mutants have been developed in many plant species, the mutagenesis efficiency varies.LD50is often used as the criterion to identify optimal treatment conditions. Many EMS-induced mutations are lethal. In order to saturate the genome, it is necessary to screen many plants to identify enough mutants for a mutant library. In this experiment, we first optimized the conditions of EMS mutagenesis in cucumber inbred line 406.Based on LD50, we found 1.5% EMS treatment for 12 h was optimal. We then treated 10 000 cucumber seeds with this mutagenic condition and obtained 631 M2lines with apparent morphological. Since not all EMS mutations exhibit visual changes, additional screening of these mutants is still needed, such as Targeting Induced Local Lesions in Genomes (TILLING) (Boualem et al. 2014; Fraenkel et al.2014). In addition to field phenotypic trait surveys, it is necessary to combine other traits such as disease resistance and fruit quality in future screening tests.

Fig. 5 Phenotypes of typical fruit mutants of M2 generation.A, wild fruit. B, shortened fruit. C, elongated fruit. D, slender fruit stalk. E, light yellow fruit peel. F, shortened fruit with dark green peel. G, shortened fruit with light green peel. H, fruit lacking warts (left for the wild type; right for the mutant). I, oval fruit. J, fruit from bisexual flower (left for the mutant; right for the wild type). K, shiny fruit surface left for the mutant; right for the wild type). L, yellow fruit pulp (above for the mutants,below for the wild type).

Table 2 Inheritance of short fruit mutation

Table 3 Inheritance of yellow-green fruit peel mutation

The inbred line 406 used as the parental line for mutagenesis in this experiment belongs to northern China ecotype and exhibits good disease resistances and high yield potentials rendering it a good breeding material.Although most mutations induced by EMS are unfavorable from a breeding perspective, some indeed showed good horticultural traits, which can be used for breeding of new cucumber varieties. For instance, fruit color and luster are important cucumber quality traits, while shiny fruit varieties are popular among the majority of consumers.While the color preference by consumers varies in different regions, breeding of wart-free varieties will be of value.Short internode length is an excellent trait for greenhouse cucumber production. Therefore, the mutation obtained from the present study with good comprehensive characters of fruit color is expected to be important parent materials for breeding. In addition, a variety of mutant materials closely related to cucumber growth and development were obtained by EMS mutagenesis from this study, including all-male plants, multiple tendrils, calyx mutations, dwarfing,bitter-free leaves, leaf color and leaf size mutations, etc.The mutants will provide an important tool for cucumber functional genomic research.

We crossed the short fruit mutant and yellow-green fruit peel mutant with the wild type to develop F2populations,respectively, and we found that both mutations were due to single recessive gene mutations. We plan to identity the casual mutations at the DNA level to understand the molecular mechanisms of these phenotypic changes. In this cucumber mutant library, for some mutated traits such as sex determination, bitter taste formation, plant dwarfism, fruit color and multi-petals, etc., the genes for them have been cloned or identified (Appendix B) (Shang et al. 2014; Zhou et al. 2015; Chen et al. 2016; Lin et al. 2016; Wang et al.2016). For example, the sex determination genes CsACS11 and CsACO2 (Chen et al. 2016) were cloned and identified with two all-male mutants obtained as the material, and the function of CsACS11 was confirmed in 2015 (Boualem et al. 2015). Both genes are related to the biosynthesis of ethylene and further confirmed the relationship between ethylene and plant sex.

5. Conclusion

In this study, we optimized the conditions for EMS mutagenesis in the cucumber inbred line 406. We found that treatment of seeds with 1.5% EMS for 12 h yielded 40% seedling survival rate and 27.9% fertility rate at the M1generation, which was effective in the construction of the cucumber EMS mutant library. Based on M2and M3data,the mutation frequency of stably inherited traits like plant architecture, leaf, floral organ and fruit traits was as high as 12.49%. Some mutations such as those exhibiting a shiny luster, lack of warts, short stems and short internodes are useful in the selection of new cucumber varieties; whereas mutations in other traits such as all-male plants, leaves lacking bitterness, multi-tendrils, or multi-petals can be of significance in functional genomics studies to advance our knowledge in basic plant biology for plant growth,development and metabolic regulation.

Acknowledgements

This study was sponsored by the National Key R&D Program of China (2016YFD0100307), the National Natural Science Foundation of China (31471871) and the Construct Program of the Key Disciplines in Hunan Province, China.

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