Research Article |
Corresponding author: Chao-Wen She ( shechaowen@aliyun.com ) Academic editor: Lorenzo Peruzzi
© 2023 Chao-Wen She, Xiang-Hui Jiang, Chun-Ping He.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
She C-W, Jiang X-H, He C-P (2023) Comparative karyotype analysis of eight Cucurbitaceae crops using fluorochrome banding and 45S rDNA-FISH. Comparative Cytogenetics 17(1): 31-58. https://doi.org/10.3897/compcytogen.v17.i1.99236
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To have an insight into the karyotype variation of eight Cucurbitaceae crops including Cucumis sativus Linnaeus, 1753, Cucumis melo Linnaeus, 1753, Citrullus lanatus (Thunberg, 1794) Matsumura et Nakai, 1916, Benincasa hispida (Thunberg, 1784) Cogniaux, 1881, Momordica charantia Linnaeus, 1753, Luffa cylindrica (Linnaeus, 1753) Roemer, 1846, Lagenaria siceraria var. hispida (Thunberg, 1783) Hara, 1948 and Cucurbita moschata Duchesne ex Poiret, 1819, well morphologically differentiated mitotic metaphase chromosomes were prepared using the enzymatic maceration and flame-drying method, and the chromosomal distribution of heterochromatin and 18S-5.8S-26S rRNA genes (45S rDNA) was investigated using sequential combined PI and DAPI (CPD) staining and fluorescence in situ hybridization (FISH) with 45S rDNA probe. Detailed karyotypes were established using the dataset of chromosome measurements, fluorochrome bands and rDNA FISH signals. Four karyotype asymmetry indices, CVCI, CVCL, MCA and Stebbins’ category, were measured to elucidate the karyological relationships among species. All the species studied had symmetrical karyotypes composed of metacentric and submetacentric or only metacentric chromosomes, but their karyotype structure can be discriminated by the scatter plot of MCA vs. CVCL. The karyological relationships among these species revealed by PCoA based on x, 2n, TCL, MCA, CVCL and CVCI was basically in agreement with the phylogenetic relationships revealed by DNA sequences. CPD staining revealed all 45S rDNA sites in all species, (peri)centromeric GC-rich heterochromatin in C. sativus, C. melo, C. lanatus, M. charantia and L. cylindrica, terminal GC-rich heterochromatin in C. sativus. DAPI counterstaining after FISH revealed pericentromeric DAPI+ heterochromatin in C. moschata. rDNA FISH detected two 45S loci in five species and five 45S loci in three species. Among these 45S loci, most were located at the terminals of chromosome arms, and a few in the proximal regions. In C. sativus, individual chromosomes can be precisely distinguished by the CPD band and 45S rDNA signal patterns, providing an easy method for chromosome identification of cucumber. The genome differentiation among these species was discussed in terms of genome size, heterochromatin, 45S rDNA site, and karyotype asymmetry based on the data of this study and previous reports.
Cucurbitaceae, cytotaxonomy, fluorescence in situ hybridization, fluorochrome banding, karyotype, karyotype asymmetry, ribosomal RNA genes (rDNA)
Cucurbitaceae, which is among the economically most important plant families, consists of about 123 genera with over 800 species distributed most in tropical and subtropical areas and very rare in temperate regions (
In higher plants, karyotype analysis has been used to characterize the genome at chromosome level, to elucidate cytotaxonomic relationships among taxa, to reveal the genetic aberrations, to understand the trends in chromosome evolution, to integrate genetic and physical maps (
In most cases, karyotyping is hampered by the lack of chromosome markers, which limits the identification of individual chromosomes. To overcome this obstacle, Giemsa and fluorochrome banding techniques as well as fluorescence in situ hybridization (FISH) technologies were successively applied in plant chromosome analysis. Double fluorochrome staining, such as CMA (chromomycin A3)/ DAPI (4,6-diamidino-2-phenylindole) staining, and PI (propidium iodide)/ DAPI staining (called CPD staining) were employed to reveal simultaneously GC-rich and AT-rich heterochromatic regions on chromosomes (
Cytogenetic studies of cucurbits started in 1920s. Earlier cytogenetic studies restricted to chromosome counting to determine the basic chromosome numbers of this family, as well as karyomorphological descriptions of some species, mainly focused on Cucumis Linnaeus, 1753 and Citrullus Schrader, 1836 (
In the current study, using the enzymatic maceration and flame-drying (EMF) method, well morphologically differentiated mitotic metaphase chromosomes of the eight Cucurbitaceae crops were prepared. The chromosomes were characterized by sequential CPD staining and FISH with 45S rDNA probe. Detailed karyotypes of these species were quantitatively constructed using the combined data of chromosome measurements, fluorochrome bands and 45S rDNA FISH signals. Four different karyotype asymmetry indices of each species were calculated for evaluating the karyological relationships among these species. The molecular cytogenetic karyotypic data were assessed to gain insights into the genome differentiation and evolutionary relationships among the eight species.
The seeds of Cucumis sativus Linnaeus, 1753, Cucumis melo Linnaeus, 1753, Citrullus lanatus (Thunberg, 1794) Matsumura et Nakai, 1916, Benincasa hispida (Thunberg, 1784) Cogniaux, 1881, Momordica charantia Linnaeus, 1753, Luffa cylindrica (Linnaeus, 1753) Roemer, 1846, Lagenaria siceraria var. hispida (Thunberg, 1783) Hara, 1948 and Cucurbita moschata Duchesne ex Poiret, 1819 were obtained from commercial seed companies in China. Cultivar accessions used in this study are described in Suppl. material
The seeds were germinated on moist filter paper in Petri dishes at 28 °C in the dark. Actively growing root tips were excised and treated in saturated α-bromonaphthalene at 28 °C for 1.0 h, and then fixed in a freshly prepared mixture of methanol and glacial acetic acid (3:1, v/v) at 4 °C, overnight. Mitotic metaphase chromosome spreads were prepared from meristem root tip cells according to
CPD staining was performed following the procedure indicated by
The probe that was used to detect the 26S-5.8S-18S rRNA gene was a 9.04-kb 45S rDNA insert from tomato (see
FISH with the 45S rDNA probe was conducted on the slides previously stained by CPD. The stained slides were washed in 2× SSC, twice for 15 min each, dehydrated in a graded ethanol series (70%, 90%, and 100%), air-dried at room temperature. Hybridization was performed as described by
Karyotype analysis followed the methodology as described by
To visualize karyotype asymmetry relationships among the eight species, bidimensional scatter diagrams for these species with MCA vs. CVCL were plotted. To determine the karyological relationships among the eight species, a principal coordinate analysis (PCoA) using Gower’s similarity coefficient were performed based on six quantitative parameters (x, 2n, TCL, MCA, CVCL, CVCI) according to the proposal by
Using the EMF method, dispersed and morphologically well differentiated mitotic metaphase chromosomes were obtained and used for karyotyping (Fig.
Mitotic chromosomes from C. sativus (A, B), C. melo (C, D), C. lanatus (E, F), B. hispida (G, H), M. charantia (I, J), L. cylindrica (K, L), L. siceraria var. hispida (M, N) and C. moschata (O, P) stained using CPD staining and sequential FISH with biotin-labelled 45S rDNA probe. A, C, E, G, I, K, M and O are the chromosomes stained using CPD. The chromosome numbers are designated by karyotyping. B, D, F, H, J, L, N and P are the chromosomes displaying 45S rDNA signals (green). The total DNA was counterstained using DAPI (blue). Scale bars: 10 µm.
Idiograms of the eight species that display the chromosome measurements, and the position and size of the fluorochrome bands and 45S rDNA FISH signals. A, B, C, D, E, F, G and H indicate C. sativus, C. melo, C. lanatus, B. hispida, M. charantia, L. cylindrica, L. siceraria var. hispida and C. moschata, respectively. The ordinate scale on the left indicates the relative length of the chromosomes (i.e. % of haploid complement). The numbers at the top indicate the serial number of chromosomes.
Species | KF | Genome size | TCL ± SE (μm) | C (μm) | RRL | CI±SE | CV CI | MCA | CV CL | St |
---|---|---|---|---|---|---|---|---|---|---|
Cucumis sativus | 2n = 14 = 12m (4SAT) + 2sm (2SAT) | 367 Mb ( |
24.36 ± 1.47 | 3.48 | 11.88–16.52 | 44.56 ± 4.94 | 11.09 | 10.88 | 12.62 | 1A |
Cucumis melo | 2n = 24 = 16m (4SAT) + 8sm | 450 Mb ( |
33.34 ± 3.21 | 2.78 | 6.87–10.72 | 39.87 ± 6.37 | 15.99 | 20.27 | 14.51 | 2A |
Citrullus lanatus | 2n = 22 = 20m + 2sm | 425 Mb ( |
24.94 ± 1.94 | 2.27 | 7.78–10.44 | 42.46 ± 3.41 | 8.03 | 15.08 | 10.92 | 1A |
Benincasa hispida | 2n = 24 = 16m (2SAT) + 8sm | 913 Mb ( |
55.93 ± 4.06 | 4.66 | 6.78–10.44 | 41.33 ± 6.20 | 14.99 | 17.33 | 12.67 | 2A |
Momordica charantia | 2n = 22 = 20m(4SAT) + 2sm | 339 Mb (Urasaki et al. 2016) | 21.31 ± 0.85 | 1.94 | 6.64–11.63 | 42.47 ± 4.60 | 10.83 | 15.06 | 17.76 | 2A |
Luffa cylindrica | 2n = 26 = 26m | 656 Mb ( |
43.75 ± 2.16 | 3.36 | 7.03–10.41 | 45.35 ± 2.73 | 6.01 | 8.86 | 9.66 | 1A |
Lagenaria siceraria var. hispida | 2n = 22 = 20m(2SAT) + 2sm | 334 Mb ( |
28.73 ± 1.69 | 2.61 | 6.67–14.52 | 41.94 ± 3.35 | 8.00 | 15.54 | 24.98 | 1B |
Cucurbita moschata | 2n = 40 = 38m + 2sm | 372 Mb ( |
38.15 ± 2.55 | 1.91 | 3.40–6.63 | 43.82 ± 3.11 | 7.11 | 12.37 | 18.61 | 2A |
The distribution of fluorochrome bands and rDNA sites in the eight Cucurbitaceae crops.
Species | Fluorochrome bands | Number (pairs) and location of 45S rDNA sites†# | |||
---|---|---|---|---|---|
Type | Distribution† | Amount (%)‡ | Band size (mean)§ | ||
Cucumis sativus | CPD | All 45S sites | 9.86 | 1.74–3.93 (2.78) | Five: 1, 3, 7S-PROX (22.06%, 12.04%, 17.41%), 2, 4L-PROX(21.53%, 15.98%) |
All CENs | 13.89 | 0.74–1.74 (1.41) | |||
1, 3, 4, 5, 7L-TERs; 4, 5, 6, 7S-TERs | 21.98 | 1.64–3.00 (2.49) | |||
Cucumis melo | CPD | All 45S sites | 6.00 | 2.42–3.59 (3.00) | Two: 1S-TER(27.03%), 2L-PROX(22.12%) |
All CENs, PCENs | 21.62 | 1.18–2.04 (1.80) | |||
Citrullus lanatus | CPD | All 45S sites | 4.52 | 2.15–2.37 (2.26) | Two: 6S-TER(41.44%), 8L-TER(48.18%) |
All CENs, PCENs | 31.25 | 2.11–3.50 (2.84) | |||
Benincasa hispida | CPD | All 45S sites | 3.77 | 1.51–2.26 (1.89) | Two: 4, 7S-TER(41.38%, 46.43%) Two: 2S-TER(27.41%), 4S |
Momordica charantia | CPD | All 45S sites | 6.95 | 3.28–3.67 (3.48) | |
All CENs, PCENs | 25.72 | 1.89–2.98 (2.34) | |||
Luffa cylindrica | CPD | All 45S sites | 6.58 | 1.03–1.83 (1.32) | Five: 1, 2, 5, 8, 12S-TER(61.27%, 71.43%, 69.42%, 70.87%, 70.19%) |
All CENs, PCENs | 25.89 | 1.70–2.21(1.99) | |||
Lagenaria siceraria var. hispida | CPD | All 45S sites | 6.77 | 1.89–4.89 (3.39) | Two: 1S-TER(15.03%), 6S-TER(47.19%) |
Cucurbita moschata | CPD | All 45S sites | 6.60 | 0.68–1.99 (1.32) | Five: 2, 3, 4, 6L-PROX(27.03%, 21.26%, 20.10%, 13.44%), 12S-PROX(15.38%) |
DAPI + | 7, 10, 11, 18-PCENs (post-FISH) | 4.88 | 0.83–1.96 (1.22) |
The diploid chromosome numbers are 2n = 2x = 14 for C. sativus, 2n = 2x = 22 for C. lanatus, M. charantia and L. siceraria var. hispida, 2n = 2x = 24 for C. melo and B. hispida, 2n = 2x = 26 for L. cylindrica, and 2n = 2x = 40 for C. moschata (Table
The karyotypes are composed of only metacentric (m) chromosomes (L. cylindrica) or metacentric and submetacentric (sm) chromosomes (the other seven species) (Table
The four different karyotype asymmetry indices are given in Table
The karyotype asymmetry relationships among the eight species that are expressed by means of bidimensional scatter plot of MCA vs. CVCL are illustrated in Figure
Bidimensional scatter plot of MCA vs. CVCL for the eight Cucurbitaceae species. C.s., C.me., C.l., B.h., M.c., L.c., L.s., and C.mo. represent C. sativus, C. melo, C. lanatus, B. hispida, M. charantia, L. cylindrica, L. siceraria var. hispida and C. moschata, respectively.
Karyological relationships among the studied species revealed by PCoA based on six karyological parameters are illustrated in Figure
PCoA for the eight Cucurbitaceae species based on x, 2n, TCL, MCA, CVCL and CVCI. C.s., C.me., C.l., B.h., M.c., L.c., L.s., and C.mo. represent C. sativus, C. melo, C. lanatus, B. hispida, M. charantia, L. cylindrica, L. siceraria var. hispida and C. moschata, respectively. PCoA1 reflects the original data characteristics before the dimensionality reduction of 60.57%. PCoA2 reflected the character of the original data before the dimensionality reduction of 23.36%. The sum of the two percentages is 83.93%, indicating that the two-dimensional coordinate system can reflect the characteristics of 83.93% of the original data.
CPD staining and DAPI counterstaining revealed distinct heterochromatin differentiation among the eight species (Figs
FISH with the 45S rDNA probe onto the chromosomes previously stained by CPD is presented in Figure
We find that all mitotic chromosomes of C. sativus can be precisely identified by the combination of the 45S rDNA FISH signals and terminal CPD bands (Figs
Precise chromosome measurement is essential for accurate karyotype analysis. Chromosomes should have morphologically distinct primary constrictions and clearly defined boundaries; otherwise, it is difficult to determine the length of chromosome arms and, consequently, to calculate chromosomal parameters (
The current karyotype of C. sativus differs in chromosome size and the classification of chromosome morphotype from some previously reported karyotypes of this species. The range of chromosome size and TCL detected in our study are similar to those of
The chromosome size of C. melo detected by us is similar to that of
The TCL of C. lanatus detected by us is larger than those of
The karyotype formula of B. hispida obtained in our study resemble those reported by
Our karyotype formula of M. charantia is similar to that reported by
The karyotype formula of L. cylindrica obtained by us is in accordance with that of
Our karyotype formula of L. siceraria var. hispida is coincident with that of
The karyotype formula of C. moschata constructed by us consists of only m and sm chromosomes, being similar to that reported by
The discrepancies in karyotype feature and 45S rDNA pattern between our results and the previous reports are probably due to differences in the accessions analyzed, the condensation level of measured chromosomes, and the difficulty in identifying chromosomes using the mitotic chromosome spreads of lower quality in the previous studies.
The total chromosome length of the haploid complement (TCL) can be used as a proxy for genome size (
The differences in CPD and DAPI+ bands, with regard to presence, position and size, reveal distinct heterochromatin differentiation among the eight cucurbits studied. CPD staining reveals the occurrence of (peri)centromeric GC-rich heterochromatin in C. sativus, C. melo, C. lanatus, M. charantia and L. cylindrica, and terminal GC-rich heterochromatin in C. sativus. (Peri)centromeric and terminal GC-rich heterochromatin was previously detected in C. sativus using CMA/DAPI staining (
The number, location and distribution of the 5S and 45S rDNA clusters in chromosomes are useful for deducing species history and phylogenetic relationship (
Concerning karyotype asymmetry is one of the most popular, cheap and widely used cytotaxonomic approach. Up to now, a variety of parameters and indices for evaluating karyotype asymmetry have been proposed, including the quali-quantitative one, Stebbins category (
In order to compare karyotypes and reconstructing karyological relationships among the eight species, we applied the methodology proposed by
The reported basic chromosome numbers of the Cucurbitaceae family ranged from x = 7 to 20, with x = 11 a prevalent number (
Detailed karyotypes of eight Cucurbitaceae crops, C. sativus, C. melo, C. lanatus, B. hispida, M. charantia, L. cylindrica, L. siceraria var. hispida and C. moschata, were reconstructed using the dataset of chromosome measurements, fluorochrome bands and 45S rDNA FISH signals. Comparative karyotyping revealed distinct variations in the karyotypic parameters, and the patterns of fluorochrome bands and 45S rDNA sites among species. The karyological relationships among the eight taxa based on six karyological parameters was basically accordant with the phylogenetic relationships revealed by DNA sequences, indicating that karyotype asymmetry study using the multivariate quantitative approach is one of the complementary characters for inferring the direction of changes of karyotype evolution in Cucurbitaceae species.
The authors have declared that no competing interests exist.
This work was supported by the Natural Science Fundation of Hunan Province, China (2019JJ40231).
Chao-Wen She https://orcid.org/0000-0003-1935-5509
Xiang-Hui Jiang https://orcid.org/0000-0003-0923-210X
The plant materials
Data type: table (docx. file)
CPD-stained mitotic metaphase chromosomes with the maximum condensation degree
Data type: figure (docx. file)
Explanation note: CPD-stained mitotic metaphase chromosomes with the maximum condensation degree from C. sativus (A), C. melo (B), C. lanatus (C), B. hispida (D), M. charantia (E), L. cylindrica (F), L. siceraria var. hispida (G) and C. moschata (H). Scale bars: 10 µm.
Chromosome measurements of the eight Cucurbitaceae crops obtained from five metaphases per species
Data type: table (docx. file)
The correlational analysis between the difference in TCL and the change in nuclear DNA content within the eight Cucurbitaceae crops using the SPSS 25.0 software
Data type: table (docx. file)
The number and position of 45S rDNA locus in Cucurbitaceae species
Data type: table (docx. file)