Repetitive DNA-based FISH enables reliable identification of individual C. clementina chromosomes
Chromosomes have long been regarded as the paramount unit of heredity carrying the genetic material of multicellular eukaryotes . Unequivocal and reproducible identification of individual chromosomes can lay a foundation for further cytological research as well as subsequent genomic and genetic studies [15, 16]. All Citrus species have relatively small chromosomes (between 2 and 4 um) with similar morphology ; as a result, differentiating chromosomes based on morphology is rather challenging [1, 27, 28].
A preliminary investigation on Citrus karyology was conducted by Krug  using an optical microscope. Later, the C-banding technique revealed that prominent heterochromatic blocks in Citrus and related genera were distinguishable in metaphase chromosomes . Subsequently, Citrus, Poncirus and Fortunella chromosomes were divided into eight different chromosomal types according to their heterochromatic CMA+/DAPE− fluorescence band variations in terms of both the numbers and distribution [26, 27, 29,30,31,32]. The combination of CMA banding and rDNA-based FISH was broadly explored to provide additional chromosome landmarks [22, 23, 25,26,27, 29, 33]. These previous cytogenetic studies detected the existence of chromosome heteromorphism in Citrus and related genera, and also identified several clusters of chromosome pairs. Nevertheless, they were unable to identify and distinguish all individual chromosome pairs [26, 31]. In this study, we reanalyzed the repetitive DNAs of Clementine. Several repetitive DNAs used as FISH probes in our study were effective cytogenetic markers that enabled unambiguous identification of individual chromosomes in Clementine. Thus, a detailed karyotype of Clementine was established using a combination of chromosome measurements and repetitive DNA element-based FISH signals (Figs. 1, 2). We are now able to present the first detailed karyotype for Clementine and demonstrate its molecular cytogenetic characterization.
Distribution characteristics of repetitive DNAs in Citrus and related genera
Many studies have shown that repetitive DNAs are functionally important for eukaryotic genomes through amplification, deletion and differentiation . Satellite DNAs comprising head-to-tail tandem repeats are believed to be the most dynamic components [17, 18], undergoing the most rapid changes in the number and position of sites within a short evolutionary period [8, 10, 34]. Here, the newly developed software package RepeatExplorer was employed for the bioinformatics analysis of repetitive elements in Clementine, as it has been extensively used to characterize repetitive elements in several plants [17, 18, 35, 36].
The failure of RepeatMasker to identify satellite repeats prompted us to reanalyze the repetitive elements in the Clementine genome . Our bioinformatics analysis showed that repetitive DNA elements constituted as much as 41.95% of the whole Clementine genome. Similar results (45%) were previously published for the Clementine genome, with different LTR-retrotransposon elements contents and numerous uncharacterized elements . One possibility might be that satellite DNA is the most challenging part of the genome to assemble . Thus, satellite elements would probably be missed or classified as other repeat elements by RepeatMasker. Here, we found that satellite DNAs are quite abundant (9.28%) in the Clementine genome. By comparison, a relatively high percentage of satellite DNAs was found in radish (Raphanus sativus L.) with 12.93%  and Tripsacum dactyloides with 14.66% , and a low percentage was found in Coix lacryma-jobi L. cultivar BJ with 0.60%, Coix aquatica Roxb. cultivar HG with 4.89% , and sunflower (Helianthus annuus L.) with 1.53% .
The distribution of repetitive DNAs may be related to karyotype characteristics and chromosomal organization. Here, CL17 was preferentially located in centromeric regions in Clementine (Fig. 1c), which dramatically increased the accuracy of determining the centromere positions of individual chromosomes and helped to characterize the features of homologous chromosomes. The telomere-associated repeat monomer 5′-TTTAGGG-3′ had a nonrandom distribution in the terminal region and tips of Clementine chromosomes (Fig. 1k), indicating that the nine homologous Clementine chromosome pairs shared the same telomere-associated repeat monomer . Further support for this conclusion came from similar patterns of telomere repeats in several other plant species [17, 18, 38]. The 5′-TTTAGGG-3′ represents the basic and canonical telomere sequence of higher plants in a chromosome-specific manner . These two repetitive DNAs are located at the centromeres and telomeres of Clementine, respectively, which potentially reflects their vital structural and functional roles in chromosome protection and nuclear organization [10, 26].
We found that 45 s rDNA sites were commonly located in the terminal regions of the short arms carrying a secondary constriction in Clementine (Figs. 1, 2). In many cases, 45 s rDNA was also observed to be restricted to nucleolus organizer regions and the telomeric regions of short arms in several other genotypes investigated here (Additional file 5). An analogous situation has also been reported in several previous studies in other Citrus species, as the activity of the 45 s rDNA genes is usually associated with nucleolus organizing regions and secondary constrictions [33, 39,40,41]. Adjacent 45 s and 5 s rDNA sites at the terminal position of the same chromosome short arm were found in the karyotype of C. clementina (Figs. 1, 2) and many other species investigated in this study (Additional file 5), indicating that 45 s and 5 s rDNA loci might be positively correlated and reflect the preferential distribution of the two sites in this arm . The FISH patterns of 45 s rDNA loci were more polymorphic than those of 5 s rDNA loci, as almost all investigated species had two 5 s rDNA loci except Mauritius papeda (C. hystrix D. C.), which had four loci (Table 2).
At the molecular level, satellite DNAs are the predominant components of heterochromatin, and are typically associated with centromeric, pericentromeric, subtelomeric and telomeric regions of chromosomes [9, 10, 18, 19, 34, 43]. The cytogenetic mapping results of the present study showed that four satellite DNAs were preferentially distributed in the subtelomeric and certain telomeric distal regions of Clementine chromosomes (Fig. 1b, d, f, h). They showed different site numbers in Clementine and other genotypes; however, favoring the terminal location can be regarded as a general tendency of their chromosomal distributions (Figs. 1, 2, Additional file 5), which is within the range of the extensively reported distribution patterns. In general, the satellite DNA sites showed much more variability both in numbers and physical chromosomal localization than did the rDNA sites in this study (Table 2). Although the reason for these preferential distributions of repetitive DNAs is not entirely known, several possible explanations have been postulated in the literature. For example, the existence of satellite DNAs without a preferential centromeric or pericentromeric distribution in the tested genotypes in Citrus and related genera possibly indicates that multiple sorts of retrotransposons comprise centromeric regions . Another potential explanation is that the terminal-preference distribution is related to the regulation of chromosome stabilization or disjunction in mitosis and meiosis [8, 10]. Furthermore, the observed trends may be related to a dynamic process [14, 31]. According to Garrido-Ramos , plant satellite DNAs are commonly structured as heterochromatin, which frequently exists in pericentromeric and subtelomeric regions.
The partially or entirely overlapping signals of the probes detected in this study are consistent with reported cases of linked 5 s–45 s rDNA sites , co-localization of the CMA+ band and 45 s rDNA sites [27, 31, 39] or satellite DNAs  in several biotypes of the subfamily Aurantioideae (Rutaceae). In previous studies, the possible reason for the linkage of these sites was assumed to be that this is the ancestral condition , while unlinked sites might reflect that these repetitive DNA elements remain mobile during species evolution [27, 33].
Implication of repetitive DNA-based FISH for the hybrid identification of Citrus and related genera
Despite the wide variety of available tools, hybrid identification still continues to be challenging in Citrus . To explore the utility of repetitive DNA-based FISH among hybrid identification, we applied these repetitive DNA probes to some presumed hybrids in Citrus species and interspecific hybrids, including citrange and Swingle citrumelo. The molecular cytogenetic method of repetitive DNA-based multicolor FISH described here enabled certain characteristic chromosomes to be identified in species and hybrids in Citrus, Poncirus, and Fortunella (Table 2, Additional file 5).
In theory, homologous chromosomes should have identical FISH distribution patterns, but here the FISH signals did not always exhibit parallel patterns in both homologous chromosomes. Conspicuously, the FISH assays of 23 investigated samples in the genus Citrus and related genera demonstrated that uneven and nonhomologous signals were commonly (Table 2, Additional file 5). This is consistent with a previous view that most citrus crops are generally characterized by highly heterozygous traits . The cytogenetic data also confirmed the widely accepted belief that many or even most Citrus species are derived from natural (spontaneous) or man-made (artificial) hybridization . For example, the asymmetrical FISH signals with an odd number of loci could certainly provide ample evidence that the Clementine is of hybrid origin, consistent with previous studies (Figs. 1, 2, Table 2). Clementine is considered to have most likely originated from a spontaneous hybrid from China and been selected by father ‘Clement’ from Algeria over a century ago [6, 44]. On the basis of morphological characteristics, Swingle has demonstrated that the Clementine is a cross between C. deliciosa Ten. and C. aurantium L. . A later serological study showed that Clementine is most closely related to ‘Baladi’ mandarin and ‘Baladi’ blood orange . Molecular markers support the assumption that Clementine is a hybrid between mandarin and sweet orange . The hypothesis of a Mediterranean mandarin × sweet orange was confirmed via single nucleotide polymorphisms (SNPs) despite one locus out of 506 suggesting incompatible genotypes . Wu et al.  investigated the conjecture at the sequence level by definitively identifying a willowleaf mandarin (C. deliciosa) and sweet orange (C. sinensis) allele at each Clementine locus. The type of cytological information obtained in our present work would be of great assistance in investigating the hybrid origin of Clementine. However, we did not confirm or discuss its putative parents. This work will be a topic of future research. However, our study allows us to conclude that heteromorphism may be an apparent indication of hybridism in Clementine. Importantly, the karyotyping analysis in our study could provide direct visual proof of the heterozygous nature of Clementine chromosomes.
The similar repetitive DNA distribution patterns in other species and hybrids within Citrus, Poncirus and Fortunella in this study are consistent with the conclusion that notable conservation exists between the three genera . An intriguing interpretation is that the three closely related genera originated from the genome of a common ancestor before speciation, which has been conserved in the genomes of Citrus plants because of a possible role of satellite DNAs in heterochromatin organization [22, 40]. Additionally, polymorphism in site numbers and variation in chromosomal locations of repetitive DNA loci is commonly observed in the three genera (Table 2). Similar results have been reported showing that Citrus chromosomes exhibit a high degree of diversity and heterozygosity, through which we can obtain further essential information to shed light upon the phylogenic and taxonomic relationships of Citrus and related genera [26, 27, 29, 33]. A plausible explanation for the observed chromosome distribution variations is the rapid amplification and/or reduction of repetitive elements [9, 34].
From the perspective of molecular cytogenetics, we found that biotypes with more signals were more ancestral, while those with fewer signals were more evolved or of hybrid origin. For example, C. hongheensis from the subgenus Papeda (Swing.) has been paid much attention in both the Swingle and Tanaka systems [2, 45]. Here, C. hongheensis showed the same number of CL1, CL2, CL3, and CL4 sites, unlike the other species. The ancient species C. mangshanensis S. W. He displayed more FISH signals than the other mandarin genotypes investigated. As mentioned above, 45 s rDNA FISH loci showed numerical variation from two to six sites, whereas 5S rDNA presented a conserved number of sites (two) in Citrus except C. hystrix (Table 2). The great similarity of C. hystrix was demonstrated by Pang et al.  with a relatively large content of polymorphic AFLP fragments and by Zhou et al.  with clustering data on its morphological characteristics, which co-contributed to the identification of its hybrid origin. The molecular cytogenetic data presented here might serve as a starting point for further elucidation of the karyotype evolution and the taxonomical relationships of Citrus, Poncirus, and Fortunella at a larger scale in the future.