1. Introduction

The Fabaceae family (Leguminosae, legume or bean family) is the third-largest flowering plant family, after the Asteraceae and Orchidaceae families ^[1][2][1,2]. It is agronomically important, as it can form a symbiotic association with nitrogen-fixing bacteria. Several species from this family serve as genetic model organisms (e.g., Medicago truncatula Gaertn. , Pisum sativum L., and Lotus japonicus L.). With more than 250 species, the clover genus, Trifolium , is one of the largest genera in this family ^[1][2][3][1,2,3]. This herbaceous genus acquired its name in reference to the characteristic form of the leaf, usually consisting of three leaflets (trifoliolate), and includes both annual and perennial species occurring natively across a large range of biotopes from meadows and open woodlands to semi-deserts and mountain ridges in temperate and, to a lesser extent, subtropical regions. The genus’s origin has been estimated to have occurred in the Early Miocene, 16–23 million years ago, and its center of origin was first assumed to be in California with its subsequent spread into Asia and hence to Europe and Africa [4]. Later, a new hypothesis was proposed of clovers originating in the Mediterranean region due to their species diversity, including the diversity in their chromosome numbers, and because the greatest occurrence of their endemic species is found in this area, with a secondary center of distribution in North America and East Africa ^[3][5][6][7][3,5,6,7]. By contrast, native clovers are absent from Australia and Southeast Asia.

Attempts have been made to divide this genus into natural groups. In the 19th century, Bossier [8] divided the genus into seven sections. A century later, eight subgenera were recognized and revised ^[1][9][1,9]. Better insight into phylogeny and the origin of the genus was facilitated by molecular analyses. These showed that Trifolium is a member of a large clade of legumes that lack one copy of the chloroplast inverted repeat ^[10][11][10,11], and a further molecular phylogenetic analysis of the internal transcribed spacer (ITS) and chloroplast genes provided evidence that most of these proposed sections are not monophyletic ^[12][13][12,13]. The most recent subgeneric classification, based on phylogenetic analyses of 218 species’ ribosomal ITS and chloroplast trnL intron sequences, was proposed by Ellison et al. [3], who divided the genus into two subgenera, Chronosemium and Trifolium , with the further subdivision of Trifolium into eight sections— Glycyrrhizum (2 species), Paramesus (2 species), Lupinaster (3 species), Trifolium (73 species), Trichocephalum (9 species), Vesicastrum (54 species), Trifoliastrum (20 species), and Involucrarium (72 species). In 2014, Trifolium phylogenetic analyses were conducted, based on highly unusual Trifolium plastomes [14].

The economic importance of this genus lies in its agricultural utilization. Historically, clovers, and especially red clovers, have been cultivated in rotation with other crops to maintain soil fertility due to their ability to establish a mutualistic relationship with root-nodulating and nitrogen-fixing bacteria. Their value was later diminished by the advent of nitrogen fertilizers, but the global need for sustainable and conservation agriculture is bringing this historical approach back into focus. Nowadays, many Trifolium species are extensively cultivated as fodder plants ( Trifolium pratense L., Trifolium repens L., Trifolium hybridum L., and Trifolium resupinatum L.), and also as green manure crops to enhance soil fertility and sustainability [15]. Further knowledge about the genomes of both wild and cultivated clovers and an understanding of their evolution will prove to be of great benefit in the future of clover breeding.

2. Chromosome Identification in Trifolium

Figure 1. Cytogenetic map of T. pratense based on the hybridization pattern of probes derived from 5S rDNA (orange circles) and 26S rDNA (pink boxes) and localization of 7 (A) or 14 (B) BAC clones corresponding to chromosome-specific microsatellite markers (green circles) (adapted from Sato et al. ^[18][43] and Kataoka et al. ^[19][68].

The easy design and production of oligonucleotide libraries has presented new opportunities to plant cytogenetics. Recently, an oligonucleotide barcode system was developed to identify all cowpea and common bean chromosomes ^[20][21][69,70]. Despite the availability of genome sequences for selected Trifolium spp., however, oligonucleotide libraries have not yet been exploited for Trifolium research.

3. Chromosomal Distribution of Ribosomal DNA Genes

Because ribosomal genes are among the best-researched regions of eukaryotic genomes, fluorescence in situ hybridization (FISH) analyses, using rDNA genes as probes, have been conducted in numerous plant species, including Trifolium. The 35S and 5S ribosomal genes are located independently in one or several loci as tandem repeats, ranging from hundreds to thousands of copies in higher vascular plant genomes ^[22][71]. While polycistronic gene 35S consists of 18S-5.8S-25S rDNA and occurs on chromosomal regions known as nucleolus organizer regions (NORs), the 5S rDNA gene is usually independent from NORs ^[23][24][72,73]. Ribosomal genes have undergone rapid evolution in their means of altering the number of copies and their localization on chromosomes ^{[25][26][27][28]}[74,75,76,77]. Therefore, rDNA genes have been proven to act as excellent cytogenetic markers for karyotype analysis, and they have been widely used to examine and understand phylogenetic relationships, chromosomal organization, and evolution in many plant species.

Roa and Guerra ^[29][30][78,79] found that, in angiosperms rDNA sites, most often number one or two 45S and one 5S per haploid genome. The localization of 45S rDNA sites was observed preferentially on the short arm and in the terminal region of chromosomes in general, but genera with predominant proximal localization were found in some families, including Fabaceae (Arachis, Lens). On the other hand, 5S rDNA localization varies in different angiosperm families. In Fabaceae, 5S rDNA is preferentially found in the proximal region.

To date, the numbers and positions of rDNA loci on chromosomes have been reported for 42 Trifolium species (Table 12; adapted from Vozárová et al. [31]). Based on ancestral state reconstruction, Vozárová et al. [31] suggested the occurrence of one 5S and one 26S locus per haploid genome separately as an ancestral condition for the whole genus. The ancestral karyotype referencing the basic chromosome number and rDNA loci constitution may resemble the karyotype of T. diffusum (Figure 2).

Figure 2. Fluorescence in situ hybridization image and schematic karyotype of T. diffusum with 5S (red) and 26S (green) rDNA probes suggested to represent the ancestral state in the Trifolium genus (adapted from Vozárová et al. [31]).

Table 12. Reported chromosome numbers 5S and 25S rRNA loci numbers in Trifolium species (adapted from Vozárová et al. [31]).

Subgenus/Section	Trifolium	Species	2n	Loci Number per 2n		Reported in
				5S	25S
CHRONOSEMIUM
	T. aureum	2x = 16	4	2	Vozárová et al. [31]
		2x = 14	4	2
	T. badium	2x = 14	2	4
			2	2
	T. campestre	2x = 14	2	2	Ansari et al. [32]	Ansari et al. [58]
	T. micranthum	2x = 16	2	2
	T. dubium	4x = 30	4	4
TRIFOLIUM
TRIFOLIUM	T. alpestre	2x = 16	10	2	Vozárová et al. [31]
			11	2
	T. arvense	2x = 14	2	2
	T. bocconei	2x = 12	2	2
	T. cherleri	2x = 10	4	10
	T. diffusum	2x = 16	2	2
	T. hirtum	2x = 10	6	2
	T. ligusticum	2x = 12	2	2
		2x = 14	2	2
	T. pallidum	2x = 16	4	2
	T. purpureum	2x = 14	2	2
	T. rubens	2x = 16	4	2
	T. squamosum	2x = 16	4	2
	T. stellatum	2x = 12	4	2
		2x = 14	4 (2w)	2
	T. pannonicum	16x = 128	16	16
	T. pratense	4x = 28	8	8	Dluhošová et al. [33]	Dluhošová et al. [80]
		2x = 14	4	5	Sato et al. [18]	Sato et al. [43]
	T. medium	8x = 64	12	8	Dluhošová et al. [33]	Dluhošová et al. [80]
TRICHOCEPHALUM	T. subterraneum subsp. subterraneum	2x = 16	2	4 (2w)	Vozárová et al. [31]
	T. subterraneumsubsp. subterraneum	2x = 16	2	2	Falistocco et al. [34]	Falistocco et al. [81]
	T. subterraneum subsp. brachycalycinum	2x = 16	2	4 (2w)
	T. israeliticum	2x = 12	10	4
VESICASTRUM	T. fragiferum	2x = 16	2	2	Vozárová et al. [31]
	T. resupinatum	2x = 16	2	2
		2x = 14	2	2
	T. spumosum	2x = 16	2	2
			4	2
TRIFOLIASTRUM	T. glomeratum	2x = 16	2	2	Vozárová et al. [31]
	T. montanum	2x = 16	2	2
	T. occidentale	2x = 16	4	2	Ansari [35]	Ansari [82]
	T. pallescens	2x = 16	2	2	Vozárová et al. [31]
	T. thalii	2x = 16	2	2
	T. repens	4x = 32	4	2	Ansari [35]	Ansari [82]
	T. uniflorum	4x = 32	4	4
	T. nigrescens subsp. nigrescens	2x = 16	2	2
	T. nigrescens subsp. petrisavii	2x = 16	2	2
	T. ambiguum	2x = 16	2	2
	T. hybridum	2x = 16	2	2
	T. isthmocarpum	2x = 16	2	6
INVOLUCRARIUM	T. chilense	2x = 16	4	2	Vozárová et al. [31]
	T. microdon	2x = 16	2	2
	T. microcephalum	2x = 16	16	16
			16	2
PARAMESUS	T. glanduliferum	2x = 16	4	2	Vozárová et al. [31]
			5 (1w)	2
			4	2
	T. strictum	2x= 14	2	2
LUPINASTER	T. lupinaster	4x = 28	8	4	Vozárová et al. [31]
		4x = 32	8	4