BIODIVERSITAS ISSN: 1412-033X Volume 24, Number 1, January 2023 E-ISSN: 2085-4722 Pages: 531-538 DOI: 10.13057/biodiv/d240162 Confirmation of mutation and genetic stability of the M4 generation of chili pepper’s (Capsicum frutescens L.) Ethyl Methane Sulfonate (EMS) mutant based on morphological, physiological and molecular characters ESTRI LARAS ARUMINGTYAS 1,♥ , ATIATURROCHMAH 1 , JONI KUSNADI 2 1 Departement of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya. Jl. Veteran, Malang 65145, East Java, Indonesia. ♥ email: laras@ub.ac.id 2 Department of Food Science and Technology, Faculty of Agricultural Technology, Universitas Brawijaya. Jl. Veteran, Malang 65145, East Java, Indonesia Manuscript received: 2 November 2022. Revision accepted: 23 January 2023. Abstract. Arumingtyas EL, Atiaturrochmah, Kusnadi J. 2023. Confirmation of mutation and genetic stability of the M4 generation of chili pepper’s (Capsicum frutescens L.) Ethyl Methane Sulfonate (EMS) mutant based on morphological, physiological and molecular characters. Biodiversitas 24: 531-538. The initial generation of Ethyl Methane Sulfonate (EMS) random mutations usually still shows high variation due to allele segregation. This research aimed to confirm the genetic differences between the M4 generation of chili pepper (Capsicum frutescens L.) mutant resulted from EMS mutations (G7/01) and the initial line (G7), and the stability of the mutant based on morphological, physiological, and molecular characters. The morphological characters and the capsaicinoid content of the mutants G7/01 with the initial line (G7) were compared. The capsaicinoid content and fruit spiciness were measured by spectrophotometer λ 280 nm. Molecular characterization was conducted using Random Amplified Polymorphic DNA (RAPD) genetic markers. Based on the morphological characters, the G7/01 mutants have some superior properties compared to the initial line G7. All the G7/01 mutant plants contain higher capsaicinoid compounds than the initial line plants. The dendrogram developed based on RAPD profile showed that all the mutant plants were positioned apart from the initial line plant, suggesting that there are some genomic changes in the mutants compared to the initial line plants. All the mutant plants, except T1, showed insignificant variation in morphological characteristic, capsaicin content, and RAPD profile, indicating genetic stability. Keywords: Capsicum frutescens, Ethyl Methane Sulfonate, mutant, RAPD, stability INTRODUCTION The genus Capsicum covers over 30 species (Tripodi and Kumar 2019). Among them, C. annuum L., C. frutescens L., C. chinense Jacq., C. pubescens Ruiz & Pav., and C. baccatum L. have been highly cultivated in the world because of their high economic value (Arumingtyas et al. 2017; Olatunji and Afolayan 2019). Capsicum frutescens, known as chili pepper, is a horticultural plant with a pungent taste caused by its capsaicin content. This species is cultivated in the tropics and sub-tropics area (Carvalho et al. 2014). In Indonesia, small-size fruit variants of C. frutescens are recognized by their local name cabai rawit, and are the most widely cultivated and used. Among many types of chili pepper found in Indonesia, six genotypes found in the markets at Malang, East Java, namely G1, G2, G3, G4, G5, and G6 (Dwinianti et al. 2018) and one genotype, G7, originated from markets in Lombok Island, Indonesia. Due to the high consumer demand for chili, various new types of chili pepper have been developed and cultivated in various regions in Indonesia. Plant breeders employed many ways. including introduction, hybridization, induced mutation, and genetic engineering to develop new superior varieties (Oladosu et al. 2016). Mutation is changing in genetic constituents that are inherited in an organism. Mutations have been reported to have a major role in increasing crop productivity and quality. More than 3,000 varieties or lines of 175 plant species have been successfully developed using mutation techniques (FAO/IAEA 2018; Mir et al. 2020). These mutants may be functional and can be used directly to meet human needs or can be a source of variation for breeding purposes. Among various types of chemical mutagens Ethyl Methane Sulfonate (EMS) is a chemical mutagen the most widely used since it is not mutagenic after being hydrolyzed. Ethyl Methane Sulfonate (EMS) is an alkylating agent which causes large changes in chromosomes, DNA, and RNA by producing a transition of GC to AT and AT to GC mutations, insertion and deletion (Wei et al. 2021). Mutations and segregation will produce genetic variation and could be expressed in subsequent offspring (Arruvitasari 2016; Oladosu et al. 2016; Wei et al. 2021). The initial generation produced by EMS mutations (M1) generally still shows high variation due to allele segregation (Arruvitasari 2016; Oladosu et al. 2016). Subsequent selection after the process of mutation induction will produce stable mutants. In semi-dwarf rice, it was reported to achieve stability at F3 after the CRISPR/Cas9-target mutation (Apriana et al. 2021).