Impaginato 289 Adv. Hort. Sci., 2023 37(3): 289­293 DOI: 10.36253/ahsc­14608 New mutations of flower shape in Nigella damascena L., its pleiotropic effects and patterns of inheritance V. Lyakh 1, 2 (*), A. Soroka 2 1 Zaporozhye National University, 66 Zhukovsky Street, 69600 Zaporozhye, Ukraine. 2 Institute of Oilseed Crops, National Academy of Agrarian Sciences of Ukraine, Institutskaya Street 1, 70417 Zaporozhye, Ukraine. Key words: flower shape, inheritance, mutant, Nigella damascena, pleiotropic effect, shortened sepal. Abstract: Two mutants with short sepals were identified after ethyl methane­ sulfonate treatment of Nigella damascena seeds. In one of them (“shs1” gene = short sepal 1), isolated from the line with double flowers, the sepals, in addi­ tion to reduced size, were divided into several rounded lobes, which granted the flower an original rose­like appearance of ornamental value. Another mutant with reduced sepals (“shs2” gene = short sepal 2) was isolated from the line with simple flowers. The allelism test showed that these two genes were non­allelic. Both mutants as pollen parents were crossed with the same line with single flowers. In a dihybrid cross, simple flower, non­reduced sepals (wild type) × double flower, reduced sepals (“shs1” gene) F1 hybrids demonstrated a wild phenotype. F2 progeny, in addition to two parental classes, showed two recombinant classes in a 9:3:3:1 ratio, indicating that flower shape and sepal size were inherited monogenously and independently, and the plant with rose­ like flowers was a double recessive homozygote. Reduced sepals (“shs2” gene) in crosses with the single flower line of wild type were inherited as a mono­ genic recessive trait, showing a 3:1 segregation ratio in F2. Both mutant genes had a number of similar pleiotropic effects, which, however, were different in strength. Thus, both mutant genes shortened leaf segments, divided the cotyle­ don leaves into several lobes, and caused disturbances in the female generative sphere, leading to a lack of seed setting. At the same time, the identification of mutants as early as at the cotyledon stage, due to the pleiotropic effect, makes it possible to select and maintain them, especially with regard to the mutant with rose­like flowers, which is highly decorative. 1. Introduction Nigella damascena L. is an annual herbaceous plant of the Buttercup family (Ranunculaceae). This is a crop of wide application, the products of which are used in medicine, food industry, and perfumery. Its seeds con­ tain about 50% fats, which consist mainly of unsaturated fatty acids, up to (*) Corresponding author: lyakh@iname.com Citation: LYAKH V., SOROKA A., 2023 ­ New mutations of flower shape in Nigella damascena L., its pleiotro‐ pic effects and patterns of inheritance. ­ Adv. Hort. Sci., 37(3): 289­293. Copyright: © 2023 Lyakh V., Soroka A. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 13 April 2023 Accepted for publication 1 August 2023 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-14608 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2023 37(3): 289­293 290 20% protein, 2­3% essential oil, enzymes, more than 20 macro­ and microelements, including essential ones (Riaz et al., 1996). Nigella is best known for the fact that nigedase is obtained from its seeds, a lipolytic enzyme prepara­ tion that is widely used in medical practice. The absence of animal protein and bile components in its composition permits to prescribe this enzyme prepa­ ration for allergies, as well as in cases where the presence of bile acids is highly undesirable. The liter­ ature contains information on the pharmacological activity of other biologically active compounds isolat­ ed from this plant, in particular, fatty oils and a num­ ber of essential oil components (Helvacıoğlu et al., 2021; Salehi et al., 2021; Benazzouz­Smail, 2023). The variety of applications of Nigella has initiated research to develop various in vitro biotechnologies for this crop from callus culture to protoplast culture (Klimek­Chodacka et al., 2020). In addition to the above, Nigella damascena has long been among the highly ornamental annual plants. Its high decorativeness is granted by rather large petal­ like sepals of white, different shades of blue, purple, pink and even red colors. In floriculture, the shape of the flower is no less valuable than the color of the flower for giving the appearance to an ornamental plant. The presence of floral dimorphism in Nigella damascena, which ensures the shape of single or dou­ ble flowers, and a variety of sepal colors allowed breeders to create a series of wonderful varieties. It has long been shown that the floral dimorphism is monogenically controlled, with the ‘single’ morph being dominant and the ‘double’ morph being reces­ sive (Toxopeus, 1927). In recent years, flower dimor­ phism and different types of petal modifications in Nigella damascena have served as the basis for using this plant as a model for elucidating the molecular control of floral dimorphism and identifying genes expressed during petal development (Jabbour et al., 2015; Zhang et al., 2020, Galipot et al., 2021). As a result of studies on induced mutagenesis in Nigella, we have identified two mutations affecting the size of the sepals, which ultimately alters the shape of the flower. These mutations, as well as their pleiotrop­ ic effects, are described in this article, which also pre­ sents the inheritance patterns for the mutant traits. 2. Materials and Methods In our studies on chemical mutagenesis, two mutations with a similar phenotypic appearance, expressed in the deformation of the sepals, were identified in Nigella damascena. In one case the mal­ formation was manifested in shortening the sepal and rounding its edge, so that the sepal instead of a pointed shape had an oval shape. It was by the pres­ ence of a shorter sepal and its rounded edge that this mutation was originally isolated. Another mutation was only designated by shortened sepals. Both muta­ tions were identified in M3 generation. The first mutant was isolated from a variety with double flow­ ers after seed treatment with ethyl methanesul­ fonate at the concentration of 0.01% and exposure for 16 hours, the other was found from a variety with single flowers as a result of seed treatment with the same mutagen at the same concentration for 6 hours. In order to check whether these two mutations are allelic, an allelism test was performed. To study the inheritance of the mutant traits, both mutants, using them as pollen parents, were crossed with the same line with single flowers. F1 hybrids were self­pollinated and in F2 families the segregation ratios were analyzed. In the cross combination “sin­ gle flower, non­reduced sepals × double flower, reduced sepals”, four classes were considered, and in the combination “single flower, non­reduced sepals × single flower, reduced sepals”, the F2 population was divided into two classes. To test if the observed frequencies of plants in F2 populations correspond the expected ones a Chi­ square test was used (Griffiths et al., 2004). 3. Results and Discussion Flowers of two mutants with deformed sepals are shown in figure 1. The flower of the mutant isolated as a result of mutagenic treatment of seeds of the double­flowered Nigella plant is shown in Figures 1a and 2b. As can be seen from the figures, the mutant, in contrast to the usual double flower (2d), was char­ acterized by shortened and more rounded sepals (“shs1” = shortened sepals with rounded edges). The incompletely opened flower of the mutant plant resembled the shape of a rose flower. In a cross combination of “single flower, non­ reduced sepals (wild type) × double flower, reduced sepals (mutant type)”, F1 hybrids had a single flower and non­reduced elongated sepals like the wild type parent. That is, a single flower completely dominated Lyakh and Soroka ‐ New mutations of flower shape in Nigella damascena 291 the double one, and non­reduced sepals over reduced ones (“shs1”). In this cross combination the parents differed by two genes and, if these genes are inherited independently, we have to obtain a typical dihybrid pattern with the four unique phenotypes in a 9:3:3:1 ratio in F2. Two F2 families showed a segre­ gation ratio where, in addition to the parental classes of single flower, non­reduced sepals (2a) and double flower, reduced sepals (2b), two recombinant classes appeared ­ single flower, reduced sepals (2c) and double flower, non­reduced sepals (2d) in approxi­ mately equal proportions (Table 1, Fig. 2). In both F2 families, there was a complete correspondence of the observed segregation ratios to the theoretically expected frequencies. The identified segregation model indicated an independent combination of flower morph and sepal shape traits and, conse­ quently, the absence of linkage between the genes that determine those traits. The flower of another mutant with deformed sepals, isolated after mutagenic treatment of seeds of a plant with simple flowers, in contrast to the first mutant, was characterized by a stronger shortening of the sepals and the absence of roundness at their ends (“shs2” = shortened sepals) (Fig. 1b). The reduc­ tion in the sepal length was accompanied by a signifi­ cant deformation of the flower pistil, which was visu­ ally revealed in the strong shortening of the sty­ loides. Some flowers of this mutant lacked them alto­ gether. Sepals reduced in length (“shs2”) in crosses with the single flower line of wild type (with non­reduced sepals) were inherited in a monogenic recessive pat­ Table 1 ­ F2 segregation for sepal shape and floral morph in cross of single flower, elongated sepals (wild type) and double flower, oval sepals (mutant type) plants in N. damascena Fig. 1 ­ Flowers of two Nigella damascena mutants with reduced sepals: a) rose­like double flower at the beginning of opening (shs1 gene); b) single flower with reduced sepa­ ls (shs2 gene). F1 phenotype Total F2 plants F2 phenotypes Segregation ratio tested χ2 (P value) single flower, non­ reduced sepals single flower, reduced sepals double flower, non­ reduced sepals double flower, reduced sepal Single flower, non­reduced sepals 100 61 17 16 6 (3:1) × (3:1) = 9:3:3:1 0.29 (0.59) Single flower, non­reduced sepals 68 40 9 13 6 (3:1) × (3:1) = 9:3:3:1 1.39 (0.24) χ205 (d.f. 3) = 7.82. Fig. 2 ­ Phenotypic classes in F2 Nigella damascena cross combi­ nations single flower, non­reduced sepals (wild type) × double flower, reduced sepals (shs1 mutant): a) single flower, non­reduced sepals; b) double flower, reduced sepals; с) single flower, reduced sepals; d) double flower, non­reduced sepals. Adv. Hort. Sci., 2023 37(3): 289­293 292 tern, showing complete dominance of the wild type over the mutant in F1, and a 3:1 segregation ratio in F2 (Table 2). The allelism test performed showed that these two genes, which determine the shortening of the sepals, are non­allelic. However, they have a number of similar pleiotropic effects. Both genes, without affecting plant height, cause shortening of true leaf segments. The bracts of both mutants are also short­ ened and more densely attached to the ripening boll than in the wild type. It should be noted that the shs2 gene as compared with the shs1 gene causes stronger changes (Fig. 3). Shortening the leaves and bracts changes the habit of the plant, making it more compact. Both mutant genes affect not only true leaves, but also cotyledons, causing them to be dissected into lobes. The division of one or two cotyledons into two lobes is characteristic of the mutant with shs2 gene (Fig. 4 b), while the multi­lobed state of both cotyle­ dons is inherent for the mutant carrying shs1 gene (Fig. 4 a). The negative effect of both mutant genes on the main function of the flower, which is reproduction, was also noted. The mutants were successfully used in various crosses as a source of pollen, but their involvement in hybridization as female parents was problematic. Sometimes such crossings were success­ ful with the shs2 mutant when using late flowers, but it was not possible to obtain seeds from the shs1 mutant even after free pollination. This indicates seri­ ous disturbances in the female generative sphere of the flowers of both mutants. Previously, in our studies with Linum grandiflorum Desf., a mutant with short petals, resembling a wild carnation flower, was identified (Lyakh, 2018). After mutagenic treatment of immature sunflower embryos, a mutant with shortened petals (ray flow­ ers) was also obtained (Soroka and Lyakh, 2009). In both cases, as for Nigella, ethyl methanesulfonate was used. The mutation identified in sunflower had a strong pleiotropic effect, affecting the stem, leaf, and even cotyledons. At the same time, true and cotyle­ don leaves had, in contrast to the elongated, round­ ed end of the leaf blade. Two­locus genetic control of petal shape was revealed in Linum grandiflorum and sunflower, where a shortened petaled plant is a double reces­ sive homozygote (Soroka and Lyakh, 2017; Lyakh, 2018). In turn, a simpler genetic system is known that controls the shape of plant organs, in particular leaves. Thus, it was found that the shape of the leaflet in cowpea is monogenously controlled, with the lanceolate leaflet shape dominant over the ovoid one (Nwofia, 2014). The same monogenic control of leaf shape, but with a co­dominant interaction of Table 2 ­ F2 segregation for sepal shape in cross of single flowered plants with non­reduced (wild type) and reduced sepals (mutant type) in N. damascena χ205 (d.f. 1) = 3.84 F1 phenotype Total F2 plants F2 phenotypes Segregation ratio tested χ2 (P value)non­reduced sepals reduced sepals Non­reduced sepals 62 51 11 3:1 1.74 (0.19) Non­reduced sepals 59 50 9 3:1 2.94 (0.09) Fig. 3 ­ Bracts and cаpsules of two Nigella damascena mutants with reduced sepals compared to the wild type: a) shs1 mutant, b) shs2 mutant, c) wild type (single flower). Fig. 4 ­ Сotyledons of two Nigella damascena mutants: a) shs1 mutant, b) shs2 mutant. Lyakh and Soroka ‐ New mutations of flower shape in Nigella damascena 293 alleles, was found in caladium (Deng and Harbaugh, 2006). As noted above, both mutations caused a partial reduction in the size of sepals of Nigella flowers. There is an opinion that the size of the flower organs is controlled by one genetic program, while the num­ ber of flower organs is determined by another genet­ ic system, independent of the first one. At the same time, they both regulate the size of the generative organ itself, the flower (Weiss et al., 2005). Our data on the independent combination of genes that deter­ mine the number of sepals and their size support the above judgment. Of the two mutations of reduced sepals identified in Nigella, only one (shs1 gene) affected the shape of the flower, turning an ordinary double flower into a rose­like flower with a decorative value. 4. Conclusions The preservation and reproduction of plants with such a flower shape for ornamental use in the usual way is problematic due to the inferiority of the female generative sphere. A partial way out of this problem could be the use of the pleiotropic effect detected at the cotyle­ don leaf stage. 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