Eggplant (Solanum melongena L.), which belongs to family Solanaceae, is a common vegetable in subtropic and tropic areas. The eggplant fruit contain abundant nutrient, such as phenolics compounds, protein, carbohydrates, mineral substance, and vitamin, which all were beneficial for human health. (San José et al., 2013). The eggplant phenolic compounds, such as anthocyanin and chlorogenic acid (CGA), have potential to scavenge reactive oxygen species. (Noda et al., 2000; Whitaker and Stommel, 2003; Hanson et al., 2006). The content of CGA in eggplant fruit were various between the eggplant cultivar and cultivated condition. The highest content of CGA in eggplant fruit was 28.0 g/kg dw as much as that in coffee (Mennella et al., 2012; Plazas et al., 2013). The delphinidin 3-rutinoside is the major form of anthocyanin found in the fruit peel which was contribution to the eggplant fruit color(Zhang et al., 2014). Nasunin which isolated from the eggplant fruit peels might be useful to prevent angiogenesis-related diseases (Matsubara et al., 2005). The eggplant has high fiber and low soluble carbohydrate content. Thus, an eggplant-based diet is recommended by the National Diabetes Education Program of NIH, Mayo Clinic, and American Diabetes Association for the management of type 2 diabetes and hypertension (Kwon et al., 2008). Otherwise, the eggplant fruit contain abundant of protein, vitamin C, mineral, dehydroascorbic acid Ayaz et al. (2015).

Although, the eggplant present diversity morphological, but the genetic of the cultivar eggplant is narrow (Meyer et al., 2012). Inducing mutations by chemical and physical methods is a highly efficient approach to increase genetic diversity (Shirasawa et al., 2016). Mutants are also potential materials for breeding new cultivar (Takagi et al., 2015). Moreover, mutants are also powerful tools for gene clone and function analysis by reverse or forward approach (Emmanuel and Levy, 2002; Takagi et al., 2013, 2015; Rizal et al., 2015).

Ethyl methane sulfonate (EMS) is one of the most popular chemical mutagens that induce mutations in plants, such as tomato (Saito et al., 2011; Shikata et al., 2016), Arabidopsis (Martin et al., 2009), and pepper (Hwang et al., 2014; Arisha et al., 2015). The EMS-induced mutants display improved traits, such as abiotic stress, phenotypic trait, and metabolite content. In rice, a salt-tolerant mutant was identified in 6,000 mutants (Takagi et al., 2015). A light-green exocarp mutant was discovered from the EMS-mutagenized cucumber line 406 with dark-green exocarp (Zhou et al., 2015). In soybean, the protein, oil, and sugar contents of the mutants are abundant (Tsuda et al., 2015). Although the EMS-induced mutation library shows abundant variations in the phenotypic trait and metabolic product content (Saito et al., 2011; Hwang et al., 2014; Arisha et al., 2015; Shikata et al., 2016), only the dominant mutation phenotypic traits are visualized in the M₁ generation. In the M₁ generation, the most identified characters are plant height, leaf color, and male sterility (Arisha et al., 2015). In the M₂ generation, the recessive character is identified in the mutational base was composition homozygosis. Thus, the most efficient time to screen the mutant by forward or reversed methods should be in the M₂ generation (McCallum et al., 2000; Takagi et al., 2013). Mutant phenotypes may not be inherited by the offspring because of DNA self-repair mechanism (Saito et al., 2011). Consequently, mutants in the M₃ or M₄ generations should be analyzed.

Next-generation sequencing is a powerful tool for analyzing the EMS-induced mutation (Gady et al., 2009; Uchida et al., 2011; Takagi et al., 2013; Henry et al., 2014). The types of EMS-induced mutation include SNV (base transition, base insertion, and base deletion), CNV, and indel section. The C/G to T/A transitions are the predominant mutations in EMS mutants (Uchida et al., 2011; Henry et al., 2014; Tsuda et al., 2015; Shirasawa et al., 2016). These mutations affect the protein synthesis or structure, thereby leading to phenotypic change. According to the base mutation effects on protein, the mutation could be divided into non-sense, frame shift, intron and intergenic, and synonymous mutations (Shirasawa et al., 2016). The whole genome sequencing result showed that intron and intergenic mutations are the predominant mutations (Shirasawa et al., 2016). Compared with the time- and labor-consuming map-based cloning methods, Mutmap technology, which is based on the mutants and whole genome sequencing, is an efficient and convenient approach in gene cloning (Takagi et al., 2013, 2015). In rice, several genes were cloned by the Mutmap methods such as hst1, (Takagi et al., 2015), ppi (Takagi et al., 2013). In cucumber, a new gene (gl2) conferring the Glabrous Trait in cucumber was identified using MutMap (Mengnan et al., 2015). The eggplant contains 24 chromosomes and the genome size was 1.13 GB. 85,446 genes were predicted in the genome which approximately 90% of the gene space was estimated (Hirakawa et al., 2014). The whole genome sequence of eggplant provides a power tool for eggplant breeding and the molecular mechanism researches.

In tomato, several mutant libraries in different backgrounds, such cultivars Micro-Tom, (Meissner et al., 1997; Saito et al., 2011), Red Setter (Gady et al., 2009), Tpaadasu (Minoia et al., 2010), and M82 (Menda et al., 2004), are available. Saito et al. (2011) created the Micro-Tom mutants and shared it (Saito et al., 2011; Shikata et al., 2016). Compared with other species of Solanaceae family, such as tomato and pepper, the mutant libraries research on eggplant is relatively limited. In our previous study, a highly homozygous inbred line E31-1 was treated by 1.0% EMS (V/V), and the physiological variations in M₁ generation were analyzed (Xiao et al., 2016). The E31-1 inbred line fruit length is about 35 cm and the color is purple. In this article, we further investigate the M₂ generation phenotypes in this EMS mutagenized line, and analyzed the CGA and anthocyanin concentrations. Our works may have considerable significance in eggplant breeding and molecular mechanisms research.

Ethyl methane sulfonate-induced mutation is a powerful tool for innovation germplasm resources. Despite the many reports about EMS-induced mutation in plant, few researches have been conducted on the EMS-induced mutation in eggplant. To innovate the germplasm resources, eggplant seed was treated with 1.0% EMS previous (Xiao et al., 2016), and the M₂ generation was analyzed in the present study. The mutants of M₂ generation showed abundant visible mutant phenotypes, such as plant height, leaf color, and fruit peel color. They also showed various content metabolites, such as CGA and anthocyanin.

In the M₂ generation, the recessive character should be present such as the yellow and albino seedling. M₂ generation showed abundant mutant phenotypes and the mutation frequency of the phenotypes were different. The plant height phenotype showed the highest mutation frequency, whereas the flower phenotype showed the lowest mutation frequency. A similar phenomenon was reported by Saito et al. (2011) and Tsuda et al. (2015). This phenomenon may be explained by the fact that the higher mutation frequency phenotypes were regulated by the more number of structure or regulatory genes. In the last decades years, the fruit size, color and shape were the mainly goals for the eggplant breeders (Kashyap et al., 2003). In the present study, some of the mutants were potential benefit for eggplant breeding, such as fruit size mutant (48-5), fruit color mutant (S9-1). Furthermore, the fruit size and color mutants are crucial to understanding the regulatory mechanisms for fruit size and color development.

Delphinidin is the major anthocyanin type in eggplant fruit peel, and it contributes to the fruit peel color (Noda et al., 2000; Matsubara et al., 2005; Zhang et al., 2014). In this study, the M₂ generation presents four types of fruit peel color, and the pH differential methods result showed that the order of anthocyanin content in the mutants was as follows: S9-1 > WT > U32-1 > L6-4. To further analyze the molecular mechanism underlying the change of fruit peel color, the known anthocyanin biosynthetic and regulatory gene (Zhang et al., 2014) expression level was detected by RT-PCR. The expression level of six biosynthesis gene, but not SmPAL, increased in S9-1 and decreased in L6-4 and U32-1 compared with WT. Zhang et al. (2014) also showed that the biosynthetic gene expression, except for SmPAL, was unregulated in the fruit peel of the purple eggplant cultivar. Those result suggested that SmPAL may not be involved in the eggplant anthocyanin biosynthetic pathway. Compared with the WT eggplant which fruit peel was black color, the expression of CHS, DFR, and ANS in a spontaneous green color mutant were significantly lower (Gisbert et al., 2016).

Several results indicated that MYB and bHLH transcriptional factor families play a vital role in the regulation of tissue color (Umemura et al., 2013; Zhu et al., 2015). Gisbert et al. (2016) showed that the regulatory genes (MybC, Myc, and Wd) was significantly decreased in the green color mutant in comparison with the WT eggplant. MYB1 positive regulates the anthocyanin accumulation in eggplant which was proved by overexpression experiment and the MYB1 upregulate most anthocyanin biosynthetic genes (Zhang et al., 2014). Meanwhile the whole genome sequence analyze result indicated two MYB-like gene may involved in the eggplant anthocyanin synthesis (Hirakawa et al., 2014). In the present study, only the SmMYB1 gene expression level increased in S9-1 and decreased in L6-4 and U32-1. To further analysis the mechanism of the different color present in the mutants, the SmMYB1 and SmbHLH gene were cloned and analyzed. The result showed that there is no mutation of SmMYB1 and SmbHLH between the WT and S9-1, L6-4 and U32-1 (Supplementary Figures S3 and S4). The result suggested that the mutational genes regulate the MYB1 and most of anthocyanin biosynthetic genes to regulate the anthocyanin accumulation. The genetic experiment result indicated that the fruit pigmentation was controlled by one or multiple genetic factors based on the material (Hirakawa et al., 2014; Gisbert et al., 2016). In our study, the L6 mutant present variegated fruit color. And the progeny of L6 line showed a different degree green color (Supplementary Figure S5). The segregation law was not compatible with Mendel inheritance models suggesting that the mutation responsible for the green color influences several genetic factors that control fruit anthocyanin accumulation.

Chlorogenic acid is beneficial to human health. The CGA content in eggplant is lower than that in coffee, and eggplant could be the CGA source in diet (Plazas et al., 2013). Compared with the results of a previous report (Plazas et al., 2014), the CGA content in the present study was relatively lower. However, the CGA content of the analyzed individual M₂ plant was higher than in WT eggplant fruit. The key genes which involved in the CGA biosynthesis was revealed (Plazas et al., 2014). The homologous gene of the biosynthetic pathway is predicated in the eggplant genome (Gramazio et al., 2014; Hirakawa et al., 2014). Nevertheless, no experiment has been conducted to confirm the genes that regulate CGA biosynthesis. The M₂ plant is not ideal breeding materials for breeding high content CGA eggplant variety, but it is a suitable material for analyzing the CGA biosynthetic mechanism in eggplant.

Despite that M₂ generation showed abundant mutations, the mutant family number was still insufficiently. Emmanuel and Levy (2002) reported that approximately 130,000 plants of fast-neutron irradiation and a range of 10³ individuals are needed for a near-saturated mutant population of tomato for reverse genetics. In future studies, the M₂ generation family should be expanded to obtain a near-saturated mutant population, and the genetic control mechanisms for the visible phenotypic changes should be analyzed.

XX-o designed the experiment and composed the manuscript. LWq analyzed the gene expression and revised the manuscript. LW and GX investigated and analyzed the phenotypic categories. LL analyzed the chlorophyll content and revised the manuscript. MF analyzed the CGA content. LY analyzed the anthocyanin content.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.