**3. Ploidy levels and chromosome structure**

Studies of ploidy levels and chromosome structure of *Capsicum* provide essential data for *Capsicum* taxonomy, assisting in the identification of cultivated, semicultivated, and wild species, as well as contributing to plant variety improvement and conservation [11].

*Capsicum* species are diploids, and most of them have 24 chromosomes (n = x = 12), but several wild species have 26 chromosomes (n = x = 13) [11, 12]. *C. annum* has 24 chromosomes; usually, two pairs are acrocentric, and 10 or 11 pairs are metacentric or submetacentric [13]. Its nuclear DNA content has 3.38 picograms (pg) per nucleus, which, in relation to other reports, ranged from 2.76 to 5.07 pg. per nucleus [14]. The chili pepper genome ranged from 1498 cm to 2268 cm and approximately two to three times larger than the tomato genome [15, 16].

In 12 *Capsicum* accessions, a chromosome number of 2n = 2x = 24 was determined, and this ploidy level is well-documented in several *Capsicum* species [17]. *Capsicum* species in the wild, such as *C. buforum,* has a ploidy level of 2n = 2x = 26 [17]. Two distinct evolutionary lines emerged throughout the history of this genus, marked by a significant separation between wild (base number x = 13) and domesticated (base number x = 12) species [17]. Multiple karyotypic formulae in the same species may occur from genetic variances within populations, which are produced by genomic responses to various environments [17]. Individuals in the same group might have different chromosomal races due to chromosomal polymorphism [17].

Plants with more karyotypic symmetry than other members of the same genus are related to those with less symmetry [18]. Even though most *Capsicum* species have 2n = 24 and have quite similar chromosomal shapes, the genus exhibits a lot of intraspecific and interspecific karyotypic diversity [18].

Karyotypic asymmetry is linked with considerable changes in TLHB and TCL across individuals of the same or nearly related species due to chromosomal modifications such as Robertsonian translocation, inversion, uneven translocations, deletions, and duplications [18]. Exposure to external elements such as climate, soil, temperature, and moisture may cause these changes [18]. *C. annum* and *C. chinense* chromosome modifications like translocations, duplications, and deletions have been identified [18]. The karyotypes of *Capsicum* in the same genus showed more genetic variability, possibly due to their high asymmetry index [19].

Studies of pepper chromosome number and morphology produce essential data for *Capsicum* taxonomy, which aid in delineating cultivated, semi-cultivated, and wild species and contribute to plant diversity conservation by providing valuable information for breeding and genetic improvement programs of this crop [20, 21].

The diploid chromosomes (2n = 2x = 24) were confirmed for each of the 12 accessions. This ploidy level is well-documented in several *Capsicum* species [19, 22–26]. For some wild *Capsicum* species, such as *C. buforum* and *C. capylopodiume,* 2n = 2x = 26 ploidy level has been reported [24]. Throughout the development of this genus, two separate lines arose, as evidenced by a clear divergence between wild (base number x = 13) and domesticated (base number x = 12) species [24]. It was hypothesized that x = 13 lines are inherited from ancestors of the x-12 plants [24].

The karyotypic formula 11M + 1SM was determined in 11 of the examined accessions, with chromosome 12 categorized as submetacentric [19]. The karyotypic formula 12M was observed in the *C. frutescens* accessions BGC 37, indicating chromosomal polymorphism compared to the other accessions [19]. For several Venezuelan accessions, the karyotypic formula 11M + 1A was reported [22]. At the same time, the formula 11M + 1A in *C. chinense* accessions was described through conventional cytogenetics in several Brazilian states [20, 27].

Genetic differences within populations, caused by the genomic response to diverse environments, might result in multiple karyotypic formulae in the same species [25]. Chromosomal polymorphism might change the karyotypic pattern of individuals in the same group, resulting in separate chromosomal races [19].

Variances in the form, size, and number of chromosomes are prevalent in populations of the same species or interspecific taxa. These differences are categorized into cytotypes or chromosomal races [19, 20]. Researchers confirmed that such variations are common in the *Capsicum* genus, whose cytotypes differ primarily in karyotypic formula and chromosomal size [19]. Secondary constrictions were found in the homologous pairs (1 and 12; 6 and 11) of the BGC 01 and BGC 37 *C. frutescens* accessions, respectively [19]. Prominent secondary constrictions were found in every *Capsicum* species, ranging from one to four per karyotype [27]. The average chromosomal size measured in various *Capsicum* species ranged from 3.29 m (BGC 49) to 7.48 m (BGC 54) [19].

Most of the *Capsicum* species have similarity (2n = 24), and the genus also exhibited intra- and interspecific karyotypic variability [28]. A higher asymmetry index across karyotypes of species in the same genus is associated with more genetic heterogeneity [20]. *Capsicum*'s chromosomal analysis at the metaphase stage revealed metacentric, submetacentric, acrocentric, or telocentric chromosomes [22, 29].

Capsicum species had symmetrical chromosomal numbers, so more extensive sampling and detailed characterization of the chromosomes, including heterochromatin distribution and sequence identification by *in situ* hybridization, must be done to distinguish between species that have the same karyotypic formula [19, 30].

Studies on pepper chromosome number and shape provide essential data for *Capsicum* taxonomy, assisting in the identification of cultivated, semi-cultivated, and wild species, as well as contributing to plant variety conservation by assisting genetic improvement efforts for this genus [31].
