Skip to main content

Ribosomal DNA localization on Lathyrus species chromosomes by FISH



Fluorescence In Situ Hybridization (FISH) played an essential role to locate the ribosomal RNA genes on the chromosomes that offered a new tool to study the chromosome structure and evolution in plant. The 45S and 5S rRNA genes are independent and localized at one or more loci per the chromosome complement, their positions along chromosomes offer useful markers for chromosome discriminations. In the current study FISH has been performed to locate 45S and 5S rRNA genes on the chromosomes of nine Lathyrus species belong to five different sections, all have chromosome number 2n=14, Lathyrus gorgoni Parl, Lathyrus hirsutus L., Lathyrus amphicarpos L., Lathyrus odoratus L., Lathyrus sphaericus Retz, Lathyrus incospicuus L, Lathyrus paranensis Burkart, Lathyrus nissolia L., and Lathyrus articulates L.


The revealed loci of 45S and 5S rDNA by FISH on metaphase chromosomes of the examined species were as follow: all of the studied species have one 45S rDNA locus and one 5S rDNA locus except L. odoratus L., L. amphicarpos L. and L. sphaericus Retz L. have two loci of 5S rDNA. Three out of the nine examined species have the loci of 45S and 5S rRNA genes on the opposite arms of the same chromosome (L. nissolia L., L. amphicarpos L., and L. incospicuus L.), while L. hirsutus L. has both loci on the same chromosome arm. The other five species showed the loci of the two types of rDNA on different chromosomes.


The detected 5S and 45S rDNA loci in Lathyrus could be used as chromosomal markers to discriminate the chromosome pairs of the examined species. FISH could discriminate only one chromosome pair out of the seven pairs in three species, in L. hirsutus L., L. nissolia L. and L. incospicuus L., and two chromosome pairs in five species, in L. paranensis Burkart, L. odoratus L., L. amphicarpos L., L. gorgoni Parl. and L. articulatus L., while it could discriminate three chromosome pairs in L. sphaericus Retz. these results could contribute into the physical genome mapping of Lathyrus species and the evolution of rDNA patterns by FISH in the coming studies in future.


Genus Lathyrus L. is one of the many genera of the family Leguminosae, genus Lathyrus includes as many as 187 species and sup-species, most of them are annual species and a few are perennial species [1], According to [2], 13 intrageneric sections have been recognized in the genus Lathyrus out of them section Lathyrus, which comprises about 30 species. Lathyrus species dispersed all over temperate regions of the northern hemisphere and spreads into tropical East Africa and South America. The main center of genus Lathyrus diversity is in the Mediterranean and Irano-Turanian regions, there are fewer centers in North and South America [2]. Members of the Lathyrus genus are used in agriculture, some as fodder crop (L. hirsutus and L. palustris), and human nutrition (L. sativus), some are grown as ornamentals, for instance, L. odoratus [3, 4]. Grass pea (L. sativus L.) is the most investigated Lathyrus species due to its importance as human consumption plant, it has survived and spread over three continents and considered one of the most resistant plant species to environmental stress and climate changes [5,6,7].

Cytologically the basic chromosome number in genus Lathyrus is x = 7, and most of the species are diploid, or allopolyploids and a few are natural autopolyploids [8,9,10,11]. Despite the stability in the chromosome number and similarity in chromosome morphology, considerable variations in chromosome size which are associated with a fourfold variation in 2C nuclear DNA amount (6.9–29.2 pg/2C) have played an important role in the evolution of Lathyrus species [12,13,14].

Fluorescence In Situ Hybridization (FISH) has made a revolution in the cytogenetic because it could explain in more detail many questions related to karyotype diversity, organization and evolution of not only individual chromosomes but also entire genomes [15,16,17,18,19,20,21,22]. Many investigations have used FISH against various taxa to distinguish specific chromosomes, or to identify individual genome in the allopolyploidy species [23,24,25,26,27,28]. In plants, FISH has been used to localize a single copy gene on its position on a specific chromosome [29,30,31], by using bacterial artificial chromosome clones (BACs) clones was useful to paint specific chromosome [32,33,34,35], to reveal chromosomal inversion [36], or a translocation between the chromosomes of the different genomes in the allopolyploid hybrids [37], it was useful in designing species-specific DNA sequences (probes) to be tested on the related species, arising in comparative cytogenetic mapping among these species [38,39,40,41].

FISH investigations using rDNA sequences as probes remain the most common, probably because the sequences are highly conserved and occur as tandemly arranged repetitive copies that vary greatly in their number among the species. Two types of rDNA (45S and 5S rDNA) are present in eukaryotes and physically they are separated from each other [42,43,44,45]. Many cytological studies have been performed on Lathyrus species to compare the karyotype of different species [10, 11, 46,47,48], or to find out the chromosome banding patterns [49,50,51,52], at the cytogenetical level FISH was very useful to localize 45S and 5S rRNA genes on different Lathyrus species [51, 53,54,55,56,57,58]. The main target of this investigation is to locate 45S and 5S rDNA on nine Lathyrus species belong to five sections by FISH.


Plant material

All of the examined species have somatic chromosome number of 2n=14 chromosomes and belong to five sections. L. gorgoni Parl (accession no. LAT. 101), L. hirsutus L. (accession no. LAT. 167), L. amphicarpos L. (accession no. LAT. 137), and L. odoratus L. (accession no. LAT. 35) belong to section Lathyrus, L. sphaericus Retz (accession no. LAT. 137) and L. incospicuus L. (accession no. LAT. 164) belong to section Linrearicarpus. The other three species belong to different sections, L. paranensis Burkart (accession no. LAT.169) belongs to section Notolathyrus, while L. nissolia L. (accession no. LAT.168) belongs to section Nissolia and L. articulatus L. (accession no. LAT. 117) belongs to section Clymenum (Table 1). The nine Lathyrus species were obtained from the germplasm collection of the Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany.

Table 1 The sections of the studied species and the chromosome no. and the arm which display 5S rDNA or 45S rDNA

Chromosome preparation:

Chromosome preparations from root tips and FISH were done according to [59] with minor modifications. Seeds were sown on two layers of moistened filter paper in a Petri dish and kept in the dark at 25°C for two days. The young germinated root tips were cut and treated with 0.02% aqueous 8-hydroxyquinoline for 3 h at 15°C and then washed several times with sterile water before fixation in freshly prepared Chloroform- acetic acid - ethanol (6:3:1) then in acetic acid - ethanol (1:3) and stored in ethanol 70%.The roots were washed in distilled water two times / 5 min and in citrate buffer (10 mM Na Citrate, pH 4.8) for 5 min, then softened in an enzyme mix [2% cellulase, and 1% pectinase (Sigma)] dissolved in the citrate buffer in an incubator at 37°C for 2 h. The root tips were washed again in citrate buffer and squashed on the slides in a drop of 45% acetic acid. The preparations were staged with a phase contrast microscope to select the slides with good separated chromosomes for further FISH experiment, then frozen on dry ice, washed with the fixation buffer and air-dried after removal of coverslips.

Fluorescence In Situ Hybridization:

The A. thaliana BAC clone T15P10 (AF167571) bearing the 45S rDNA sequence was labelled with digoxigenin by nick translation, and the 5S rDNA probe was amplified from genomic DNA of A. thaliana and labelled with biotin by PCR with primers specific for the coding region [60]. The biotinylated 5S rDNA was detected by avidin~Texas Red (Vector Laboratories) and amplified by biotinylated goat anti-avidin (Vector Laboratories) and avidin~Texas Red. Digoxigenin-labelled probes were detected by mouse anti-digoxigenin (Jackson ImmunoResearch Laboratories) and goat anti-mouse antibodies conjugated with Alexa 488 (Molecular Probes). The chromosomes were counterstained with DAPI (2 μg/ml). The images were captured with a Zeiss Axioplan 2 epifluorescence microscope equipped with a Spot 2e CCD camera. Images were pseudo-coloured and merged using Adobe Photoshop CS software (Adobe). The karyotypes of the studied species have been done manually from the images which have been taken by the epifluorescence microscope. The available chromosome images were magnified in Adobe Photoshop CS software (Adobe) to enlarge the image to the size in which the difference in chromosome size could be identified, then each chromosome was copied and pasted separately and arranging the number of these chromosomes according to their decreasing in the size, taking in account the homologous chromosomes which bear the 45S and 5S rRNA genes.


FISH has been performed to locate 45S and 5S rRNA genes on the chromosomes of nine Lathyrus species all have 2n=14, and belong to five sections.

The loci of 45S (in green) and 5S rDNA (in red) probes as revealed by double-target FISH on metaphase chromosomes preparations of the nine examined species have been shown in Fig. 1, and their karyotypes are shown in Fig. 2, and Table 1 summarizes the chromosome number and arm which display the 5S and 45S rDNA loci.

Fig. 1
figure 1

Mitotic metaphase chromosomes of nine Lathyrus species after FISH with rDNA probes, 45S rDNA probe was detected by FITC (green signals), and 5S rDNA probe by Texas red (red signals). The chromosomes were counterstained with DAPI. Bar = 5μ. a) L. paranensis Burkart b) L. nissolia L. c) L. odoratus L. d) L. hirsutus L. e) L. amphicarpos L. f) L. gorgoni Parl. g) L. articulatus L. h) L. sphaericus Retz i) L. incospicuus L.

Fig. 2
figure 2

The Karyotypes of the examined Lathyrus species with 5S rDNA (in red) and 45S rDNA (in green) Bar = 5μ. a) L. paranensis Burkart b) L. nissolia L. c) L. odoratus L. d) L. hirsutus L. e) L. amphicarpos L. f) L. gorgoni Parl. g) L. articulatus L. h) L. sphaericus Retz i) L. incospicuus L.

The detected 45S and 5S rRNA gene loci using double-FISH experiment on the metaphase chromosomes of each of studied species as follows:

L. paranensis Burkart (Fig. 1a) belongs to section Notolathyrus, showed one interstitial 5S rRNA gene locus on the short arm of the chromosome no. 3 and one 45S rRNA gene locus on the long arm of chromosome no. 4.

L. nissolia L. (Fig. 1b) belongs to section Nissolia, exhibited one interstitial locus of 5S rRNA and one 45S rRNA gene locus on the opposite arms of chromosome no. 1.

L. odoratus L. belong to section Lathyrus, has one stretched interstitial 45S rRNA gene site on chromosome no. 4, which is distinguished with a big satellite, while two 5Sr RNA gene loci were observed in this species, one interstitial and one distal 5S rDNA on the short arm of chromosome pair no. 3 (Fig.1c).

L. hirsutus L. belongs to section Lathyrus too, exhibited a single terminal 45S rDNA site, and one proximal 5S rDNA site located on the short arm of chromosome pair no. 3 (Fig.1d).

L. amphicarpos L. the third studied species belongs to section Lathyrus, it has a big terminal 45Sr RNA gene locus on the long arm of the largest chromosome and interstitial 5Sr RNA gene locus on the short arm of the same chromosome (no. 1), in addition to another distal 5Sr RNA gene locus on the short arm of chromosome no. 2 (Fig. 1e).

L. gorgoni Parl is the fourth studied species belongs to section Lathyrus, it was characterized by having one large stretched interstitial 5S rDNA site on the short arm of middle-sized chromosome pair (no.5), while the 45S rDNA site was at a distal position on the short arm of large chromosome pair no. 3 (Fig.1f).

L. articulatus L. belongs to section Clymenum, it also exhibited one large stretched proximal 45S rDNA site on the long arm of chromosome pair no. 4, whereas the 5S rDNA site was located on the middle of the short arm of chromosome no. 1 (Fig. 1g).

L. sphaericus Retz belongs to section Linrearicarpus. it was characterized by having two proximal 5S rRNA gene loci on the short arm of chromosomes no. 2 and no. 3, and one big terminal 45Sr RNA gene locus on the long arm of chromosome no. 4 (Fig. 1h)

L. incospicuus L. belongs to section Linrearicarpus too, this species was characterized by having one distal 5S rRNA gene locus on the short arm of longest arm chromosome (no. 1), and exhibited one large stretched distal 45S rDNA site on the long arm of the same chromosome pair as well (Fig. 1i).


Genus Lathyrus includes 187 species and sup-species, some of which have economic importance as food, fodder, or ornamental crops. Lathyrus species are distributed in the regions of the Northern Hemisphere and outspread into tropical South America East Africa [1, 16]. Most Lathyrus species are diploid (2n = 14). [2] classified genus Lathyrus depending on morphological traits into 13 intrageneric sections. Section Nissolia is monotypic has only one species L. nissolia. While section Aphaca is ditypic has two species L. aphaca and L. stenolobus, and section Lathyrus comprises about 30 species. Despite the stability in chromosome number, many investigations mentioned a variation in chromosome features, e.g. size, centromere position, and the size, number and position of secondary constrictions [11, 48, 52, 61, 62].

Lathyrus species show uniform chromosome morphology which is reflected in a homogeneous karyotype arrangement [9, 49]. Nevertheless, others have observed interspecific karyotype variations allowing species identifications [53, 61]. Such divergences have been also detected at the infraspecific level, especially in the extensively studied L. odoratus L. and L. sativus L. [53, 63, 64]. Many cytological studies have been performed to find out the chromosome banding patterns and karyotypes and /or ideograms of some Lathyrus species including L. odoratus, L. articulatus and L. incospicuus L. [49,50,51,52, 56], in previous investigation, the chromosome measurements, karyotype and chromosome banding patterns of six out of the nine studied species (L. gorgoni Parl, L. hirsutus L., L. amphicarpos L., L. sphaericus Retz, L. paranensis Burkart, and L. nissolia L.) have been studied by [52]. At the cytogenetical level, FISH was very useful to localize 45S and 5S rRNA genes on other Lathyrus species as well [51, 53,54,55,56,57,58].

Despite of few literatures stated the localization of rRNA gene loci by FISH in three of the examined species (L. odoratus L, L. paranensis Burkart and L. hirsutus L.), no previous FISH investigations have been found on the other six species. In L. odoratus, significate differences in its karyotype have been reported often to differ in the position and number of secondary constrictions, in the current study it was observed that L. odoratus (accession no. LAT. 35 ) has only one stretched interstitial 45S rRNA gene site on chromosome no. 4, which is distinguished with a big satellite. In the study of [63] they described cultivars with up to eight secondary constrictions, whereas [64] reported some with none at all. In the study of [53], silver staining (binds to the NOR, the nucleoli, and sometimes shows a tendency to bind chromosome centromere in some taxa, and dependent on the transcription rRNA genes) and in situ hybridization (independent of transcription and may also detect non-functional rDNA sites) were used to identify the nucleolar organizer regions (NORs) among six Lathyrus species, they noticed that L. odoratus and L. hirsutus were very similar with a large and a small sub-metacentric pair and five pairs of acrocentrics, and in both species the largest of the acrocentrics had a secondary constriction very close to the telomere of the short arm, in the same investigation they mentioned that L. hirsutus had a single pair of silver positive terminal spots. In L. odoratus, there was staining at or near the centromere in addition to staining at the secondary constrictions, but it was clear that the cells of L. odoratus have two pairs of terminal silver positive regions at the ends of two of the largest acrocentric pairs, and in the same study, the rDNA loci represented by FISH reflected the same number of signals in these two species.

The current study is in agreement with the result of [53] only with regard to L. hirsutus L., where it exhibited a single terminal 45S rDNA locus on the short arm of chromosome pair no. 3 and one proximal 5S rDNA site located in the middle of the same arm in this study, but with regard to L. odoratus L. disagrees. [46] used Silver staining as well to recognize the nucleolar organizer regions (NORs) in L. odoratus and they mentioned that there were four-terminal NORs on the short arms of pairs 4 and 5 with active ribosomal genes. However, L. odoratus L. bears microsatellites in two pairs of chromosomes (nos. 3 and 5) via the karyotype analysis by [11, 57] studied 14 species of Lathyrus (included L. odoratus L. and L. paranensis Burkart) by double FISH to determine the 45S and the 5S rDNA loci in addition to the CMA and DAPI banding patterns, they analyzed too the karyotypes in relation to geographic and climatic changes. In their investigation, they detected two loci of 45S rDNA on the short arm of chromosomes no. 4 and no. 5 in L. odoratus L., and they stated in L. paranensis one 5S rDNA locus and one 45S rDNA locus on separate chromosomes, which in agreement with the obtained result in the present study with regard to L. paranensis L., but disagrees with regard to L. odoratus L.

Nuclear DNA content may vary from 6.9–29.2 pg/2C [12,13,14] measured the nuclear DNA content (1C) in many plant genera, among them the genus Lathyrus, according to their measurements, there was no correlation between the genome size and the number or position of rDNA loci, nevertheless depending on the DNA content measurements (pg /1C) within section Lathyrus they recorded that L. amphicarpos L. and L. gorgoni Parl. were closely related (4.80, 5.80 pg/1C, respectively), and L. hirsutus L. (10.00 pg/1C) and L. odoratus L. (7.80 pg/1C) relatively similar too. In a former study [65] the DNA contents of different Lathyrus species have been measured by flow cytometry, among them L. gorgoni Parl (11.5 pg/2C), L. hirsutus L. (12.7 pg/2C) and L. odoratus L. (14.3 pg/2C), and the gradually increasing in the genome size in these three species is in agreement with their relationships as revealed by FISH in the current investigation.

The differences in genome size generally in plants and correspondingly in Lathyrus could be attributed first to the variations in the chromosome complements size and to the non-coding elements such as transposable elements, pseudogenes, and other repetitive sequences throughout the chromosome structure. The obtained banding patterns from many investigations supported the non-randomness of genomic change in Lathyrus as well, because the species mostly have uniform karyotypes and their banding patterns with similar quantity and base composition [11, 50]. Therefore, it is better to focus on the role of rDNA as repetitive sequences, rather than as coding gene loci and number [66] stated a slightly positive correlation between genome size and rDNA copy number in a restricted number of the eukaryotic test groups, and in some Lathyrus species by [51]. On the other hand [67] claimed against the existence of a precise relationship between the two parameters. The investigations of [68, 69] within the same taxonomic groups were also unsuccessful to reach this consensus, which in agreement with the obtained result of this study. The investigations of [70,71,72,73,74] informed that there is no correlation between rDNA copy number and genome size, and this correlation still a mystery because both characters are dynamic and are subjected for some changing during short periods of time. The chromosomal numbers, locations and structure of the 5S and 45S ribosomal DNAs (rDNA) in plants are nowadays available online [74, 75].

FISH results of the investigated species in this study were summarized in Table 1, and Fig. 3. shows the dendrogram (UPGMA, suing Jaccard’s coefficient) which reflects the relationships among the studied species by Past program (Paleontological statistics software package for education and data analysis, [76]), which showed that the conventional plant taxonomy at the section level in the studied Lathyrus species is partially reflected by the number and loci of both rRNA genes. This is in agreement with the study of [77] based on amplified fragment length polymorphism (AFLP), where they found that L. gorgoni and L. hirsutus (section Lathyrus) grouped in same cluster, whereas L. articulatus (section Clymenum) was related to L. inconspicuous (section Linearicarpus), while L. inconspicuous and L. sphaericus (section Linearicarpus) were placed on distinct branches, and the results of [78] based on the internal transcribed spacer plus 5.8S-coding region of nuclear ribosomal DNA and cpDNA sequence data supported this. These relationships were partially reflected by the investigation of [79], who studied the relationships of different sections of genus Lathyrus depending on the cpDNA restriction site, and their obtained phylogenetic suggestions were used to test some species of genus Lathyrus, including L. hirsutus L. and L. odoratus L. which have been grouped under the same clade, while L. gorgoni Parl and L. amphicarpos L., under another clade, and L. nissolia L with L. sphaericus Retz in separate clade, whereas L. paranensis Burkart was found to be distantly related.

Fig. 3
figure 3

Dendrogram of the studied Lathyrus species based on the number and loci of 5S and 45S rDNA

Double-target FISH by utilizing 5S and 45S rDNA loci as probes in the present study was helpful to discriminate the chromosomes of each of the nine studied species, which possibly could be used as chromosome markers. FISH could discriminate only one chromosome pair out of the seven pairs in three species, in L. hirsutus L., L. nissolia L. and L. incospicuus, and two chromosome pairs in five species, in L. paranensis Burkart, L. odoratus L., L. amphicarpos L., L. gorgoni Parl. and L. articulatus L., while it could discriminate three chromosome pairs in L. sphaericus Retz. these results could contribute into the physical genome mapping of Lathyrus species and the evolution of rDNA patterns by FISH in the coming studies in future.


Physical mapping of 5S and 45S rDNA loci by FISH on the chromosomes of the nine Lathyrus species could be used as chromosomal markers to discriminate the chromosome pairs of each of the examined species. FISH could discriminate only one chromosome pair out of the seven pairs in three species, in L. hirsutus L., L. nissolia L. and L. incospicuus, and two chromosome pairs in five species, in L. paranensis Burkart, L. odoratus L., L. amphicarpos L., L. gorgoni Parl. and L. articulatus L., while it could discriminate three chromosome pairs in L. sphaericus Retz. The conventional taxonomy at the section level in the studied Lathyrus species could not be proved by the number or loci of rRNA genes.

Availability of data and materials

Authors declare that all generated and analyzed data are included in the article. All plant materials (different Lathyrus species seeds) were identified and collected in Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany.



Fluorescence in situ Hybridization


nucleolar organizing regions




Ribosomal DNA


Ribosomal RNA


chloroplast DNA


Amplified Fragment Length Polymorphism


  1. Allkin R, Goyder DJ, Bisby FA, White RJ (1986) Names and synonyms of species and subspecies in the Vicieae. Vicieae Database Project. 7:1–75.

  2. Kupicha FK (1983) The infrageneric structure of Lathyrus. Notes from the Royal Botanic Garden Edinburgh. 41:209–244.

  3. Kenicer GJ, Kajita T, Pennington RT, Murata J (2005) Systematics and biogeography of Lathyrus (Leguminosae) based on internal transcribed spacer and cpDNA sequence data. Am J Bot 9:1199–1209.

    Article  Google Scholar 

  4. Vaz Patto MC, Rubiales D (2014) Lathyrus diversity: available resources with relevance to crop improvement-L. sativus and L. cicera as case studies. Annals of botany 113: 895–908.

  5. Almeida NF, Leitão ST, Krezdorn N, Rotter B, Winter P, Rubiales D, Vaz Patto MC (2014b) Allelic diversity in the transcriptomes of contrasting rust-infected genotypes of Lathyrus sativus, a lasting resource for smart breeding. BMC Plant Biol 14:376 https://doi. org/ 10.1186/s12870-014-0376-2

  6. Almeida NF, Krezdorn N, Rotter B, Winter P, Rubiales D, Vaz Patto MC (2015) Lathyrus sativus transcriptome resistance response to Ascochyta lathyri investigated by deep Super SAGE analysis. Front Plant Sci 6:178

    Article  Google Scholar 

  7. Lambein F, Travella S, Kuo Y et al (2019) Grass pea (Lathyrus sativus L.): orphan crop, nutraceutical, or just plain food? Planta 250:821–838

    Article  Google Scholar 

  8. Gutiérrez J, Vaquero F, Vences F (1994) Allopolyploid vs. autopolyploid origins in the genus Lathyrus (Leguminosae). Heredity 73: 29–40. 10.1038/ hdy. 1994.95

  9. Klamt A, Schifino‐Wittmann MT  (2000) Karyotype morphology and evolution in some Lathyrus (Fabaceae) species of southern Brazil. Genetics and Molecular Biology 23:463–467.

  10. Seijo JG, Fernández A (2001) Cytogenetic analysis in Lathyrus japonicus Willd. (Leguminosae). Caryologia 66:173–179.

    Article  Google Scholar 

  11. Seijo JG, Fernández A (2003) Karyotype analysis and chromosome evolution in South American species of Lathyrus (Leguminosae). Am J Bot 90:980–987 10. 3732/ajb.90.7.980

    Article  Google Scholar 

  12. Narayan RKJ, Rees H (1976) Nuclear DNA variation in Lathyrus. Chromosoma 54:141–154

    Article  Google Scholar 

  13. Bennett MD, Smith JB (1991) Nuclear DNA amounts in angiosperms. Philos. Trans. R. Soc. Lond. B Biol. Sci. No. 334:309–345

    Article  Google Scholar 

  14. Bennett MD, Leitch IJ (2012) Plant DNA C-values database (release 6.0, Dec. 2012). Accessed 5 Jan 2013

  15. Hizume M, Shiraishi H, Matsusaki Y, Shibata F (2013) Localization of 45S and 5S rDNA on Chromosomes of Nigella damascena, Ranunculaceae. Cytologia 78: 379–381. 10.1508/cytologia.78.379

  16. Ikeda K, Sato S, Matoba H, Nagano K, Uchiyama H (2013) Molecular Cytogenetic Analysis of the Critically Endangered Trigonotis radicans var. radicans and var. sericea and Allied Species in Japan. Cytologia 78:297-303.

  17. Kuroki Y, Shibata F, Hizume M (2013) Chromosome Bandings and Signal Pattern of FISH Using rDNAs in Bellevalia romana. Cytologia 78:399–401.

    Article  Google Scholar 

  18. Howe ES, Murphy S, Bass HW (2013) Three-Dimensional Acrylamide Fluorescence In Situ Hybridization for Plant Cells. In: Pawlowski W., Grelon M, Armstrong S (eds) Plant Meiosis. Methods in Molecular Biology (Methods and Protocols), vol 990. Humana Press, Totowa, NJ. 10.1007/978-1-62703-333-6_6

  19. Yokomi I, Ogiwara H, Kohno T, Yokota J, Satoh H (2013) Comparative fiber-FISH reveals what happened at the integration site of the transfected plasmid DNA. Cytologia 78:121–122.

    Article  Google Scholar 

  20. Lakshmanan PS, Van Laere K, Eeckhaut T, Van Huylenbroeck J, Van Bockstaele E, Khrustaleva L (2015) Karyotype analysis and visualization of 45S rRNA genes using fluorescence in situ hybridization in aroids (Araceae). Comp Cytogenet 9:145‐160.

    Article  Google Scholar 

  21. Dechyeva D, Schmidt T (2016) Fluorescent In Situ Hybridization on Extended Chromatin Fibers for High-Resolution Analysis of Plant Chromosomes. In: Kianian S, Kianian P (eds) Plant Cytogenetics. Methods in Molecular Biology, vol 1429. Humana Press, New York, NY. 10.1007/978-1-4939-3622-9_3

  22. Jiang J (2019) Fluorescence In Situ Hybridization in plants: recent developments and future applications. Chromosome Res 27:153–165.

    Article  Google Scholar 

  23. Jiang J, Gill BS (1994) Nonisotopic in situ hybridization and plant genome mapping: the first 10 years. Genome 37:717–725.

    Article  Google Scholar 

  24. Jiang J, Gill BS (2006) Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research. Genome 49:1057–1068.

    Article  Google Scholar 

  25. Robledo G, Lavia GI, Seijo G (2009) Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection. Theor Appl Genet 118:1295–1307.

    Article  Google Scholar 

  26. Iwata A, Greenland CM, Jackson SA (2013) Cytogenetics of Legumes in the Phaseoloid Clade. Plant Genome 6.

  27. Ortiz AM, Robledo G, Seijo G, Valls JFM, Lavia GI (2017) Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section Rhizomatosae and related diploid species (Arachis, Leguminosae). J Plant Res 130:791-807. 10.1007 /s10265-017-0949-x

  28. Van-Lume B, Mata-Sucre Y, Báez M, Ribeiro T, Huettel B, Gagnon E, Leitch IJ, Pedrosa-Harand A, Lewis GP, Souza G (2019) Evolutionary convergence or homology? Comparative cytogenomics of Caesalpinia group species (Leguminosae) reveals diversification in the pericentromeric heterochromatic composition. Planta Dec 250:2173-2186. 10.1007/s00425-019-03287-z

  29. Danilova TV, Friebe B, Gill BS (2012) Single-copy gene fluorescence In Situ Hybridization and genome analysis: Acc-2 loci mark evolutionary chromosomal rearrangements in wheat. Chromosoma 121:597–611.

    Article  Google Scholar 

  30. Danilova TV, Friebe B, Gill BS (2014) Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae. Theor Appl Genet 127:715–730.

    Article  Google Scholar 

  31. Dillon A, Varanasi VK, Danilova TV, Koo DH, Nakka S, Peterson DE, Tranel PJ, Friebe B, Gill BS, Jugulam M (2017) Physical mapping of amplified copies of the 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant Amaranthus tuberculatus. Plant Physiol 173:1226–1234.

    Article  Google Scholar 

  32. Lysak MA, Fransz PF, Ali HBM, Schubert I (2001) Chromosome painting in Arabidopsis thaliana. The Plant Journal 28:689–697.

    Article  Google Scholar 

  33. Gu YQ, Ma Y, Huo N, Vogel JP, You FM et al (2009) A BAC-based physical map of Brachypodium distachyon and its comparative analysis with rice and wheat. BMC genomics 10:496.

    Article  Google Scholar 

  34. Han YH, Zhang T, Thammapichai P, Weng YQ, Jiang JM (2015) Chromosome-specific painting in Cucumis species using bulked oligonucleotides. Genetics 200:771–779.

    Article  Google Scholar 

  35. Albert PS, Zhang T, Semrau K, Rouillard J-M, Kao Y-H, Wang C-JR, Danilova TV, Jiang JM, Birchler JA (2019) Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships. Proc Natl Acad Sci U S A 116:1679–1685.

    Article  Google Scholar 

  36. Fransz P, Linc G, Lee C‐R, Aflitos SA, Lasky JR, Toomajian C, Ali H, Peters J, van Dam P, Ji X, Kuzak M, Gerats T, Schubert I, Schneeberger K, Colot V, Martienssen R, Koornneef M, Nordborg M, Juenger TE, de Jong H, Schranz ME (2016) Molecular, genetic and evolutionary analysis of a paracentric inversion in Arabidopsis thaliana. Plant J 88:159–178.

    Article  Google Scholar 

  37. Ali HBM, Fransz, P, Schubert I (2000a) Localization of 5S RNA Genes on Tobacco Chromosomes. Chromosome Res 8: 85–87.

  38. Doganlar S, Frary A, Daunay MC, Lester RN, Tanksley SD (2002) A comparative genetic linkage map of eggplant (Solanum melongena) and its implications for genome evolution in the Solanaceae. Genetics 161:1697–1711.

  39. Lysak MA, Lexer C (2006) Towards the era of comparative evolutionary genomics in Brassicaceae. Pl Syst Evol 259:175–198.

    Article  Google Scholar 

  40. Betekhtin A, Jenkins G, Hasterok R (2014) Reconstructing the Evolution of Brachypodium Genomes Using Comparative Chromosome Painting. PloS one, 9(12), e115108.

  41. Braz GT, He L, Zhao H, Zhang T, Semrau K, Rouillard JM, Torres GA, Jiang JM (2018) Comparative oligo-FISH mapping: an efficient and powerful methodology to reveal karyotypic and chromosomal evolution. Genetics 208:513–523.

    Article  Google Scholar 

  42. Hemleben V, Zentgraf U (1994) Structural Organization and Regulation of Transcription by RNA Polymerase I of Plant Nuclear Ribosomal RNA Genes. In: Nover L (ed) Plant Promoters and Transcription Factors. Results and Problems in Cell Differentiation (A Series of Topical Volumes in Developmental Biology), vol 20. Springer, Berlin, Heidelberg.

    Chapter  Google Scholar 

  43. Cabral-de-Mello DC, Oliveira SG, de Moura RC, Martins C (2011) Chromosomal organization of the 18S and 5S rRNAs and histone H3 genes in Scarabaeinae coleopterans: insights into the evolutionary dynamics of multigene families and heterochromatin. BMC Genet. 12:88.

    Article  Google Scholar 

  44. JS H‐H, Schwarzacher T (2011) Organization of the plant genome in chromosomes. The Plant Journal 66:18–33.

    Article  Google Scholar 

  45. Mondin M, Aguiar-Perecin ML (2011) Heterochromatin patterns and ribosomal DNA loci distribution in diploid and polyploid Crotalaria species (Leguminosae, Papilionoideae), and inferences on karyotype evolution. Genome. Sep; 54:718-26.

  46. Battistin A, Biondo E, Coelho LGM (1999) Chromosomal characterization of three native and one cultivated species of Lathyrus L. in Southern Brazil. Genet Mol Biol 22:557–563.

  47. Arzani A (2006) Karyotype study in some Lathyrus L. accession of Iran. Iran J Sci Technol 30:9–17

    Google Scholar 

  48. Badr SF (2007) Karyotype Analysis and Chromosome Evolution in Species of Lathyrus (Fabaceae). Pakist J  Biol Sci 10:49–56.

  49. Lavania UC, Sharma AK (1980) Giemsa C Banding in Lathyrus L. Botanical Gazette 141:199–203.

    Article  Google Scholar 

  50. Ünal F, Wallace AJ, Callow RS (1995) Diverse heterochromatin in Lathyrus. Caryologia 48:47–63.

    Article  Google Scholar 

  51. Ali HBM, Meister A, Schubert I (2000b) DNA content, rDNA loci, and DAPI bands reflect the phylogenetic distance between Lathyrus species. Genome 43: 1027-1032. 10.1139/g00-070

  52. Ali HBM, Abd Elhady EA, Barakat HM (2005) DAPI-banding patterns in six Lathyrus species. Egypt J Genet Cytol 34:267–279

    Google Scholar 

  53. Murray B, Bennett M, Hammett K (1992) Secondary constrictions and NORs of Lathyrus investigated by silver staining and in-situ hybridization. Heredity 68:473–478.

    Article  Google Scholar 

  54. Nandini A.V., Cytogenetics and interspecific hybridization in Lathyrus L., Ph.D. thesis, The University of Auckland, New Zealand 1997. https://researchspace.auckland. /uoa-docs/rights.htm

  55. Ceccarelli M, Ceccarelli M, Sarri V, Polizzi E, Andreozzi G, Cionini PG (2010) Characterization, Evolution and Chromosomal Distribution of Two Satellite DNA Sequence Families in Lathyrus species. Cytogenet Genome Res 128:236–244.

    Article  Google Scholar 

  56. Chalup L, Grabiele M, Neffa VS et al (2012) Structural karyotypic variability and polyploidy in natural populations of the South American Lathyrus nervosus Lam. (Fabaceae). Plant Syst Evol 298:761–773.

    Article  Google Scholar 

  57. Chalup L, Samoluk SS, Neffa VS et al (2015) Karyotype characterization and evolution in South American species of Lathyrus (Notolathyrus, Leguminosae) evidenced by heterochromatin and rDNA mapping. J Plant Res 128:893–908

    Article  Google Scholar 

  58. Murray BG (2016) The 2016 Banks Memorial Lecture: Cytogenetics and ornamental plant breeding: An ongoing partnership. New Zealand Garden Journal 19:14–18

    Google Scholar 

  59. Fransz P, Armstrong S, Alonso-Blanco C, Fischer TC, Torres-Ruiz RA, Jones G (1998) Cytogenetics for the model system Arabidopsis thaliana. Plant J. 13: 867-876. 10.1046/j.1365-313X.1998.00086.x

  60. Gottlob-McHugh SG, Lévesque M, MacKenzie K, Olson M, Yarosh O, Johnson DA (1990) Organization of the 5S rRNA genes in the soybean Glycine max (L.) Merrill and conservation of the 5S rDNA repeat structure in higher plants. Genome 33:486–494.

    Article  Google Scholar 

  61. Battistin A, Fernández A (1994) Karyotypes of four species of South America natives and one cultivated species of Lathyrus L. Caryologia 47:325–330.

    Article  Google Scholar 

  62. De KK, Pal TK, Mondal A, Majumder M, Ghorai A (2018) Extended centromere and chromosomal mosaicism in some varieties of grass pea, Lathyrus sativus L. The Nucleus 62:21–30.

    Article  Google Scholar 

  63. Sharma AK, Datta PC (1959) Application of improved technique in tracing karyotype differences between strains of Lathyrus odoratus L. Cytologia 24:389–402.

    Article  Google Scholar 

  64. Fouzaar A, Tandon SL (1975) Cytotaxonomic investigations in the genus Lathyrus. Nucleus 18:24–44.

  65. Nandini AV, Murray BG, O’Brien IEW, Hammett KRW (1997) Intra- and interspecific variation in genome size in Lathyrus (Leguminosae). Botanical Journal of the Linnean Society 125(4):359–366.

    Article  Google Scholar 

  66. Ingle J, Timmis JN, Sinclair J (1975) The relationship between satellite deoxyribonucleic acid, ribosomal ribonucleic acid gene redundancy, and genome size in plants. Plant Phys 55:496–501.

    Article  Google Scholar 

  67. Gall JG (1981) Chromosome structure and the C-value paradox. J. Cell Biol 91:3s–14s.

    Article  Google Scholar 

  68. Bobola MS, Smith DE, Klein AS (1992) Five major nuclear ribosomal repeats represent a large and variable fraction of the genomic DNA of Picea rubens and P. mariana. Mol Biol Evol 9: 125–137.

  69. Lloyd AD, Mellerowicz EJ, Riding R, Little CHA (1996) Changes in nuclear genome size and relative ribosomal gene content in cambial region cells of Abies balsamea shoots during the development of dormancy. Can J Bot 74:290–298.

    Article  Google Scholar 

  70. Široký J, Lysák MA, Doležel J et al. (2001) Heterogeneity of rDNA distribution and genome size in Silene spp. Chromosome Res 9:387–393.

  71. Prokopowich CD, Gregory TR, Crease TJ (2003) The correlation between rDNA copy number and genome size in eukaryotes. Genome 46(1):48–50 10.1139 /g02-103

    Article  Google Scholar 

  72. Kolano B, Siwinska D, McCann J, Weiss-Schneeweiss H (2015) The evolution of genome size and rDNA in diploid species of Chenopodium s.l. (Amaranthaceae). Botanical J. of the Linnean Society 179(2):218–235.

  73. Hoang PTN, Schubert V, Meister A et al. (2019) Variation in genome size, cell and nucleus volume, chromosome number and rDNA loci among duckweeds. Sci Rep 9, 3234.

  74. Garcia S, Garnatje T, Kovařík A (2012) Plant rDNA database: ribosomal DNA loci information goes online. Chromosoma 121:389–394.

    Article  Google Scholar 

  75. Garcia S, Kovařík A, Leitch AR, Garnatje T (2017) Cytogenetic features of rRNA genes across land plants: analysis of the Plant rDNA database. Plant J. 89:1020–1030.

    Article  Google Scholar 

  76. Hammer Ø, Harper DAT, Ryan PD (2001) PAST: Paleontological Statistics Software Package for Education and Data Analysis. Paleontología Electrónica 4(1):1–9

    Google Scholar 

  77. Badr A, El Shazly H, El Rabey H, E. Watson L (2002) Systematic relationships in Lathyrus sect. Lathyrus (Fabaceae) based on amplified fragment length polymorphism (AFLP) data. Can J Bot (80):962–969.

  78. Kenicer GJ, Kajita T, Pennington RT, Murata J (2005) Systematics and biogeography of Lathyrus (Leguminosae) based on internal transcribed spacer and cpDNA sequence data. Am J Bot 92:1199–1209.

  79. Asmussen CB, Liston A (1998) Chloroplast DNA Characters, Phylogeny, and Classification of Lathyrus (Fabaceae). Am J of Bot 85:387–401.

Download references


The authors would like to thank the Genetics and Cytology department, National Research Centre, Dokki, Giza, Egypt, for performing the experiment in its laboratory. The authors are grateful to all the researchers whom we cited in this review for their significant and valuable research.


The Research experiment was partially sponsored by Department of Genetics and Cytology, National Research Centre, Cairo, Egypt

Author information

Authors and Affiliations



HA performed the FISH experiment part. HA and SO wrote the manuscript, participated in the data discussion, data analyses, and drafting of the manuscript, all authors have read and approved the manuscript.

Corresponding author

Correspondence to Hoda B. M. Ali.

Ethics declarations

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ali, H.B.M., Osman, S.A. Ribosomal DNA localization on Lathyrus species chromosomes by FISH. J Genet Eng Biotechnol 18, 63 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: