Advances in the Molecular Cytogenetics of Bananas, Family Musaceae

Abstract

The banana is a staple food crop and represents an important trade commodity for millions of people living in tropical and subtropical countries. The most important edible banana clones originated from natural crosses between diploid Musa balbisiana and various subspecies of M. acuminata. It is worth mentioning that evolution and speciation in the Musaceae family were accompanied by large-scale chromosome structural changes, indicating possible reasons for lower fertility or complete sterility of these vegetatively propagated clones. Chromosomal changes, often accompanied by changes in genome size, are one of the driving forces underlying speciation in plants. They can clarify the genomic constitution of edible bananas and shed light on their origin and on diversification processes in members of the Musaceae family. This article reviews the development of molecular cytogenetic approaches, ranging from classical fluorescence in situ hybridization (FISH) using common cytogenetic markers to oligo painting FISH. We discuss differences in genome size and chromosome number across the Musaceae family in addition to the development of new chromosome-specific cytogenetic probes and their use in genome structure and comparative karyotype analysis. The impact of these methodological advances on our knowledge of Musa genome evolution at the chromosomal level is demonstrated. In addition to citing published results, we include our own new unpublished results and outline future applications of molecular cytogenetics in banana research.

Keywords: BAC clones; DNA repeats; chromosomes; flow cytometry; fluorescence in situ hybridization; karyotyping; oligo painting; rRNA genes.

Figure 1

Estimation of nuclear genome size and chromosome number in Musella lasiocarpa. (A) Histogram of relative nuclear DNA content obtained after flow cytometric analysis of propidium iodide-stained G1 nuclei of M. lasiocarpa (2C = 1.174 pg) and Glycine max (2C = 2.5 pg), which served as the internal reference standard. Measurements were performed according to Bartoš et al. [20]. (B) mitotic metaphase chromosomes of M. lasiocarpa (2n = 2x = 18). The chromosomes were counterstained with DAPI (blue); the probe specific to 45S rDNA sequence (red) was localized to secondary constrictions. Probe specific to 45S rDNA was prepared according to Čížková et al. [8]. Bars correspond to 5 µm.

Figure 2

Determination of Musa ploidy level by flow cytometry. Histograms of relative nuclear DNA content obtained after flow cytometric analysis of DAPI-stained nuclei isolated from various accessions of Musa, and chicken red blood cell nuclei (CRBC) served as the internal reference standard. (A) triploid accession ‘Green Red’ (genomic constitution AAA; peak ratio 0.75); (B) triploid accession ‘Ternate’ (genomic constitution AAB; peak ratio 0.76); (C) diploid M. acuminata ‘Hawain 2′ (genomic constitution AA; peak ratio 0.51); (D) tetraploid accession ‘CRBP 15′ (genomic constitution AAAB; peak ratio 1). Measurements were performed according to Christelová et al. [9].

Figure 3

Phylogenetic tree of Musaceae family members. The tree is constructed from ITS1–ITS2 sequence regions of ribosomal DNA using the BioNJ method implemented in SeaView [24]. Names of the four sections of Musa, species belonging to the corresponding section, and their basic chromosome number (x) are printed in different colors: Eumusa section (red), resp. (blue), Rhodochlamys section (orange), Callimusa section (pink), and Australimusa section (green). In addition, M. ingens from the Ingentimusa section is included in the figure (purple).

Figure 4

Mitotic chromosomes of genus Musa. Mitotic metaphase plates of (A) Musa acuminata ssp. burmannicoides ITC0249 (2n = 2x = 22) from Eumusa section; (B) M. laterita ITC1575 (2n = 2x = 22) from Rhodochlamys section; (C) M. maclayi ITC1207 (2n = 2x = 20) from Australimusa section; and (D) M. beccarii ITC1070 (2n = 2x = 18) from Callimusa section. The chromosomes were stained with DAPI, and the images are shown in white pseudo-color. Arrows indicate secondary constrictions. One secondary constriction broke from the rest of the chromosome as shown in (D). Bars correspond to 5 µm.

Figure 5

Examples of the genomic distribution of DNA satellites, BAC clone, LINE element, and rDNA as determined in mitotic metaphase chromosomes of diploid Musa accessions after FISH. Satellite repeat sites CL18 and CL33 probe hybridization are marked by long and thick arrows, respectively. (A) CL18 (red) and CL33 (green) on ‘Long Tavoy’ chromosomes; (B) 5S rDNA (red) and CL18 (green) on ‘Maia Oa’ chromosomes; (C) 5S rDNA (red) and CL33 (green) ‘Long Tavoy’ chromosomes; (D) CL18 (red) and LINE element (green) on ‘Tuu Gia’ chromosomes; (E) 5S rDNA (red) and CL18 (green) on ‘Pisang Klutuk Wulung’ chromosomes; (F) BAC clone 2G17 (red) and 5S rDNA (green) on chromosomes of ‘Cameroun’; (G) BAC clone 2G17 (red) and CL18 (green) on ‘Tani’ chromosomes; (H) 45S rDNA (red) and 5S rDNA (green) on M. schizocarpa ITC 0560 chromosomes; (I) 5S rDNA (red) and CL33 (green) on M. schizocarpa ITC 1002 chromosomes; (J) CL18 (red) and CL33 (green) on M. schizocarpa ITC 1002 chromosomes; (K) CL18 (red) and 5S rDNA (green) on M. schizocarpa ITC 1002 chromosomes. Chromosomes were counterstained with DAPI (blue). Bars correspond to 5 µm. Retrieved from Čížková et al. [8].

Figure 6

Oligo painting FISH on meiotic pachytene chromosome spreads of Musa. (A) M. acuminata ssp. malaccensis ‘Pahang’ (2n = 2x = 22, AA; chromosome 1 in red, chromosome 4 in green); (B) M. balbisiana ‘Tani’ (2n = 2x = 22, BB; chromosome 1 in red, chromosome 3 in green). Chromosomes were counterstained with DAPI (blue). Arrows point to the region translocated from chromosome 3 to chromosome 1 in M. balbisiana. Bars correspond to 10 µm. Retrieved from Šimoníková et al. [54].

Figure 7

Oligo painting FISH on mitotic metaphase plates of two Musa accessions (A) M. acuminata ssp. zebrina ‘Maia Oa’ (2n = 2x = 22, AA; long arm of chromosome 3 in red, short arm of chromosome 8S green; (B) aneuploid East African Highland banana (EAHB) clone ‘Imbogo’ (2n = 3x − 1 = 32, AAA; long arm of chromosome 3 in green, short arm of chromosome 8 in red; (C) idiogram of M. acuminata ssp. zebrina ‘Maia Oa’; (D) idiogram of aneuploid East African Highland banana (EAHB) clone ‘Imbogo’. Chromosomes were counterstained with DAPI (blue). Bars correspond to 5 µm. Idiograms were retrieved from Šimoníková et al. [89].

Figure 8

FISH on mitotic metaphase plates of two Musa interspecific hybrids. (A) Hybrid ‘Sar’ ITC1213 (2n = 3x = 32, AAT; Radka5 DNA sequence (red); (B) Hybrid ‘Umbubu’ ITC0854 (2n = 2x = 21, AT; Radka6 DNA sequence (green). Chromosomes were counterstained with DAPI (blue). Bars correspond to 5 µm.