Background and Objective: Ajowan (Carum copticum L.) is one of the important annual spice and medicinal plants cultivated in various regions of the world, particularly in the Middle East, Iran, and India. Fennel (Foeniculum vulgare Mill.) is also an important aromatic and medicinal plant and is among Iran’s export commodities. Fennel's seeds are the major essential oil-producing organ. Both species belong to the family Apiaceae. In recent years, with the cultivation and domestication of indigenous medicinal plant populations in Iran, several superior genotypes with high yield and economic cultivation potential have been identified and are currently at the stage of registration and release. In the introduction of new cultivars, yield, phytochemical, molecular, and cytogenetic characteristics should be investigated and documented as components of cultivar identification. The simultaneous use of molecular markers and cytogenetic characteristics can facilitate the identification and discrimination of genotypes at different genetic levels and provide complementary information on genetic diversity and chromosomal structure. Given the economic and medicinal importance of these two species and the need for accurate identification of superior genotypes, simultaneous assessment of molecular diversity and cytogenetic characteristics can contribute to completing the identification profiles of these genotypes. Therefore, this study aimed to develop molecular profiles using SCoT markers, document the chromosome number, evaluate karyotype characteristics, and ploidy variation of superior ajwaan and fennel
Materials and Methods: Three ajowan populations, including Sarbisheh (gene bank code 37251), Shiraz (12313), and Khorasan (1085), and two fennel populations, including Ardabil (10589) and Isfahan (945), were investigated. For molecular diversity analysis, genomic DNA was extracted from young leaves and amplified using six Start Codon Targeted (SCoT) markers. PCR products were separated by electrophoresis on 2% agarose gels, and bands were scored as a binary matrix based on their presence or absence. Primer performance was evaluated using Polymorphic Information Content (PIC), Effective Multiplex Ratio (EMR), Resolving Power (RP), and Marker Index (MI). In addition, genetic diversity indices, including observed number of alleles (Na), effective number of alleles (Ne), Shannon’s information index (I), expected heterozygosity (He), and unbiased expected heterozygosity (uHe), were estimated, and genetic
similarity among populations was determined based on Nei’s genetic similarity. For cytogenetic analysis, young roots were subjected to pretreatment, fixation, hydrolysis, and staining, and chromosome preparations were obtained using the squash method. Suitable metaphase plates were identified using a light microscope and photographed with a digital camera. Chromosome number, chromosome dimensions, and karyotypic symmetry indices were then measured and compared.
Results: In the molecular analysis of fennel populations using six SCoT markers, a total of 60 bands were detected, of which 56 (93.33%) were polymorphic. The highest values of PIC, EMR, RP, and MI were 0.422, 15, 10, and 6.33, respectively, obtained from SCoT11. Based on the genetic diversity indices Na, Ne, I, He, and uHe, the Isfahan population (945) of fennel exhibited higher genetic diversity than the Ardabil population (10589), and Nei’s genetic similarity between the two populations was 0.783. In ajowan, 50 bands were detected, of which 45 (90%) were polymorphic. The highest values of PIC, EMR, RP, and MI were 0.401, 16, 9.55, and 6.42, respectively, obtained from SCoT20. The highest and lowest genetic similarity were observed between the Sarbisheh and Shiraz populations (0.876) and between the Sarbisheh and Khorasan populations (0.757), respectively. Among the ajowan populations, Shiraz (12313) showed the highest genetic diversity, whereas Sarbisheh (37251) exhibited the lowest. Cytogenetic analysis revealed chromosome numbers of 2n=18 in ajowan and 2n=22 in fennel populations. In ajowan, chromosome length ranged from 2.29 to 2.34, the centromeric index ranged from 0.348 to 0.371, and the symmetry index ranged from 72.190 to 76.460. In fennel, chromosome length was 2.954 and 3.009 in the Ardabil and Isfahan populations, respectively, while the centromeric index was 0.391 and 0.350, and the symmetry index was 66.056 and 65.481, respectively. Despite these numerical differences, statistical analyses revealed no significant differences in chromosome traits or karyotypic symmetry indices among the populations of either species.
Conclusion: The findings demonstrated that SCoT markers were highly effective in detecting polymorphism and genetic differentiation among the superior populations of ajowan and fennel. SCoT11 in fennel and SCoT20 in ajowan exhibited the highest discriminatory efficiency and may therefore be useful for the preliminary screening of genotypes. In contrast, the relative stability of karyotypic characteristics indicated that the observed molecular differences were not necessarily accompanied by major chromosomal changes. Therefore, the integration of molecular and cytogenetic data can provide a suitable basis for genetic fingerprinting, genotype identification, and the management and conservation of genetic resources in these medicinal plants. |