Research Article |
Corresponding author: Chao-Wen She ( shechaowen@aliyun.com ) Academic editor: Viktoria Shneyer
© 2017 Chao-Wen She, Lin Wei, Xiang-Hui Jiang.
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 CW, Wei L, Jiang XH (2017) Molecular cytogenetic characterization and comparison of the two cultivated Canavalia species (Fabaceae). Comparative Cytogenetics 11(4): 579-600. https://doi.org/10.3897/compcytogen.v11i4.13604
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The two cultivated Canavalia (Adanson, 1763) species, Canavalia gladiata (N. J. von Jacquin, 1788) A. P. de Candolle, 1825 and Canavalia ensiformis (Linnaeus, 1753) A. P. de Candolle, 1825 are closely related based on morphological and molecular phylogenetic data. However, the similarities and differences in genome organization between them have not been evaluated at molecular cytogenetic level. Here, detailed karyotypes of both species were constructed using combined PI and DAPI (CPD) staining, rDNA-FISH and self-genomic in situ hybridization (sGISH). For further comparison, comparative genomic in situ hybridization (cGISH) and sequence analysis of 5S rDNA were applied. Their chromosomes were accurately identified by sGISH and rDNA-FISH signals. Both species had the karyotype formula 2n = 22 = 18m + 4m-SAT, but the karyotype of C. ensiformis was shorter and more asymmetric than that of C. gladiata. They displayed similar CPD bands at all 45S rDNA sites and centromeres. C. gladiata had ten centromeric 5S rDNA loci and two SC (secondary constriction)-associated 45S rDNA loci. C. ensiformis had nine centromeric and one interstitial 5S loci, two SC-associated and one proximal 45S loci. Their sGISH signal patterns displayed both basic similarities and distinct differences. Reciprocal cGISH generated prominent signals in all pericentromeric regions and 45S sites. There was lower level of sequence identity of the non-transcribed spacer between their 5S rDNA repeats. These data confirmed the evolutionary closeness between C. gladiata and C. ensiformis and demonstrated obvious differentiation between their genomes, and supported the opinion that C. ensiformis is more advanced in evolution than C. gladiata.
Canavalia cultivars, karyotype, 5S rDNA, 45S rDNA, fluorochrome banding, in situ hybridization
The genus Canavalia Adanson, 1763, belonging to the tribe Diocleae of the family Fabaceae, comprises about sixty pantropical species (
Although C. gladiata and C. ensiformis differ in geographical origin, they are closely related. This fact was established by their highly similar morphologies and seed proteins (
Detailed karyotypes displaying chromosome morphology, heterochromatin distribution, and location of repetitive DNA sequences and bacterial artificial chromosome (BAC) have been constructed for many plant species. These are used to reveal chromosome-level genome organization, investigate the evolutionary relationships among related species, and integrate genetic and physical maps (
Combined propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI) staining (CPD staining; a type of fluorochrome banding) simultaneously reveals GC- and AT-rich chromosome regions (
The GISH technique, a modification of FISH using genomic DNA as a probe, is conventionally utilized for identifying parental genomes in hybrids and allopolyploids (
In the present study, molecular cytogenetic characterization of C. gladiata and C. ensiformis was performed using sequential CPD staining, dual color FISH with 5S and 45S rDNA probes, and sGISH. Detailed karyotypes of the two species were established using a combination of chromosome measurements, CPD bands, and rDNA-FISH and sGISH signals. cGISH of the genomic DNA of one species to the chromosomes of the other species was also performed. The 5S rDNA repeats of the two species were cloned, sequenced, and mapped using FISH. The data were assessed to gain insights into the evolutionary relationships between the two cultivated Canavalia species.
Seeds of C. gladiata (Jacq.) DC. were obtained from the Chinese Crop Germplasm Resources Information System (CGRIS) and collected in China. Seeds of C. ensiformis (L.) DC. were kindly provided by the United States (US) National Plant Germplasm System (NPGS) and collected in Brazil (PI 337078). For GISH and amplification of the 5S rDNA sequences, genomic DNA (gDNA) was extracted from young leaves using cetyltrimethylammonium bromide (CTAB) based on the method described by
The 5S rDNA sequences (including the coding regions and NTS) were amplified by polymerase chain reaction (PCR) using the specific primers 5S1 (5' -GGATGGGTGACCTCCCGGGAAGTCC-3') and 5S2 (5' -CGCTTAACTGCGGAGTTCTGATGGG-3') deduced from the 5S rRNA gene coding sequence of Beta vulgaris Linnaeus, 1753 (
The procedure for mitotic chromosome preparation was essentially the same as that reported in published protocols (
The CPD staining followed the procedure described in
A 45S rDNA clone containing a 9.04-kb tomato 45S rDNA insert (
FISH with 5S and 45S rDNA probes and cGISH were carried out after CPD staining on the same slides. FISH with cloned 5S rDNA repeats and sGISH were conducted on the slides that were previously stained with CPD and hybridized with the 5S and 45S rDNA probes. The slides were then washed in 2× SSC (Saline-sodium citrate buffer) twice for 15 min each, dehydrated through an ethanol series (70%, 90%, and 100%, 5 min each), and used for hybridization. The in situ hybridization procedure followed the protocol described in detail by
For each species, five metaphase plates that had been subjected to sequential CPD staining, rDNA-FISH, and sGISH were measured using Adobe Photoshop version 8.01 to obtain chromosome relative lengths (RL; percentage of haploid complement), arm ratios (AR; long arm/short arm), fluorochrome band and sGISH signal sizes, and percent distance from the centromere to the rDNA site (di = d × 100/a; where d = distance from the middle of the rDNA sites to the centromere; a = corresponding chromosome arm length). The satellite length was included in the respective chromosome arm length. The stretched secondary constriction (SC) lengths were omitted. The total haploid complement length (TCL; the karyotype length) was measured using the five metaphase cells with the highest degree of chromosome condensation. The arm ratios were used to classify the chromosomes according to the system described by
For both species, genomic DNA amplification produced one major fragment of approximately 950 bp and one minor fragment of approximately 450 bp. Amplicons were cloned. Ten from each transformation were screened to verify the presence of the insert. Five clones of each fragment were sequenced.
Sequence analysis showed that all inserts correspond to 5S rDNA repeats. Each fragment was neighbored by 40 bp and 58 bp of the gene at the 5' and the 3' ends, respectively (Fig.
Alignment of the major fragments amplified from the 5S rDNA repeats of Canavalia gladiata (C. g.) and Canavalia ensiformis (C. e.). The entire 120-bp 5S rRNA gene and the 40 and 58 bp of the gene flanking the 5' and 3' ends are enclosed in a box; the intragenic promoter motifs are underlined.
Representative mitotic chromosomes of C. gladiata and C. ensiformis are shown in Figure
Chromosome measurements of Canavalia gladiata (C.g.) and Canavalia ensiformis (C.e.) obtained from five metaphases per species.
Species | Chr. No. | Relative length (%) | Arm ratio ± SD | Type | Centromeric CPD band size‡ ± SD | sGISH signal size‡ | ||||
Short arm ± SD | Long arm ± SD | Total ± SD | Short arm ± SD | Long arm ± SD | Total ± SD | |||||
C. g. | 1 | 5.25 ± 0.23 | 6.70 ± 0.24 | 11.95 ± 0.41 | 1.28 ± 0.05 | m | 1.53 ± 0.27 | 3.19 ± 0.19 | 4.17 ± 0.13 | 7.35 ± 0.22 |
2 | 4.43 ± 0.10 | 6.39 ± 0.36 | 10.82 ± 0.31 | 1.44 ± 0.10 | m | 1.93 ± 0.15 | 3.07 ± 0.33 | 2.96 ± 0.19 | 6.03 ± 0.38 | |
3 | 4.72 ± 0.37 | 5.69 ± 0.19 | 10.40 ± 0.28 | 1.21 ± 0.12 | m | 1.63 ± 0.17 | 1.44 ± 0.28 | 3.56 ± 0.13 | 5.00 ± 0.28 | |
4 | 4.63 ± 0.20 | 5.57 ± 0.17 | 10.20 ± 0.17 | 1.21 ± 0.08 | m | 1.67 ± 0.14 | 3.19 ± 0.22 | 2.50 ± 0.15 | 5.69 ± 0.09 | |
5 | 3.61 ± 0.08 | 5.53 ± 0.09 | 9.15 ± 0.16 | 1.53 ± 0.02 | m | 1.76 ± 0.19 | 3.61 ± 0.08 | 1.94 ± 0.24 | 5.55 ± 0.31 | |
6 | 3.71 ± 0.46 | 5.11 ± 0.22 | 8.83 ± 0.61 | 1.39 ± 0.16 | m† | 1.54 ± 0.10 | 3.71 ± 0.46 | 2.79 ± 0.26 | 6.50 ± 0.67 | |
7 | 4.14 ± 0.20 | 4.50 ± 0.26 | 8.64 ± 0.38 | 1.09 ± 0.06 | m† | 1.14 ±0.18 | 4.14 ± 0.20 | 2.13 ± 0.32 | 6.27 ± 0.50 | |
8 | 3.42 ± 0.25 | 4.68 ± 0.12 | 8.11 ± 0.21 | 1.38 ± 0.13 | m | 1.93 ± 0.27 | 2.33 ± 0.23 | 3.01 ± 0.14 | 5.34 ± 0.31 | |
9 | 3.20 ± 0.08 | 4.53 ± 0.20 | 7.73 ± 0.18 | 1.42 ± 0.08 | m | 1.79 ± 0.11 | 3.20 ± 0.08 | 1.77 ± 0.21 | 4.97 ± 0.25 | |
10 | 3.39 ± 0.10 | 4.31 ± 0.29 | 7.70 ± 0.38 | 1.27 ± 0.06 | m | 1.36 ± 0.16 | 1.62 ± 0.28 | 2.55 ± 0.32 | 4.18 ± 0.31 | |
11 | 2.44 ± 0.16 | 4.03 ± 0.21 | 6.47 ± 0.27 | 1.65 ± 0.13 | m | 1.32 ± 0.12 | 2.43 ± 0.18 | 1.73 ± 0.17 | 4.15 ± 0.33 | |
Total | 42.96 ± 0.51 | 57.04 ± 0.51 | 100 | 17.59 ± 1.13 | 31.93 ± 0.33 | 29.11 ± 0.32 | 61.04 ± 0.19 | |||
C. e. | 1 | 5.58 ± 0.23 | 7.02 ± 0.38 | 12.60 ± 0.18 | 1.26 ± 0.11 | m | 2.54 ±0.57 | 3.18 ± 0.17 | 3.99 ±0.29 | 7.17 ±0.36 |
2 | 4.43 ± 0.18 | 6.62 ± 0.28 | 11.05 ± 0.38 | 1.50 ± 0.07 | m | 2.32 ±0.63 | 2.53 ± 0.29 | 3.14 ±0.20 | 5.66 ±0.40 | |
3 | 5.02 ± 0.16 | 5.33 ± 0.60 | 10.35 ± 0.51 | 1.06 ± 0.14 | m | 2.14 ±0.68 | 3.77 ± 0.24 | 2.32 ±0.11 | 6.10 ±0.29 | |
4 | 4.68 ± 0.16 | 5.35 ± 0.43 | 10.03 ± 0.53 | 1.14 ± 0.08 | m | 1.93 ±0.40 | 1.67 ± 0.21 | 3.57 ±0.19 | 5.24 ±0.37 | |
5 | 3.49 ± 0.19 | 5.36 ± 0.28 | 8.85 ± 0.29 | 1.54 ± 0.13 | m | 1.80 ±0.23 | 2.12 ± 0.17 | 2.13 ±0.32 | 4.25 ±0.31 | |
6 | 3.96 ± 0.29 | 4.80 ± 0.25 | 8.76 ± 0.46 | 1.22 ± 0.08 | m† | 2.08 ±0.51 | 3.96 ± 0.29 | 2.87 ±0.24 | 6.83 ±0.31 | |
7 | 3.65 ± 0.25 | 4.46 ± 0.16 | 8.11 ± 0.15 | 1.23 ± 0.13 | m† | 1.45±0.18 | 3.65 ± 0.25 | 2.48 ±0.55 | 6.13 ±0.57 | |
8 | 3.76 ± 0.25 | 4.25 ± 0.28 | 8.01 ± 0.41 | 1.13 ± 0.10 | m | 1.88 ±0.33 | 1.65 ± 0.22 | 2.73 ±0.15 | 4.38 ±0.34 | |
9 | 2.99 ± 0.23 | 4.73 ± 0.32 | 7.72 ± 0.23 | 1.59 ± 0.22 | m | 1.76 ±0.20 | 2.99 ± 0.23 | 1.81 ±0.19 | 4.80 ±0.35 | |
10 | 3.35 ± 0.24 | 4.34 ± 0.22 | 7.69 ± 0.44 | 1.30 ± 0.05 | m | 1.67 ±0.18 | 2.06 ± 0.15 | 2.56 ±0.26 | 4.62 ±0.37 | |
11 | 2.59 ± 0.27 | 4.23 ± 0.19 | 6.82 ± 0.44 | 1.64 ±0.12 | m | 1.68 ±0.08 | 2.59 ± 0.27 | 1.76 ±0.38 | 4.35 ±0.61 | |
Total | 43.50 ± 0.76 | 56.50 ± 0.76 | 100 | 21.24 ± 3.11 | 30.18 ± 1.36 | 29.35 ± 2.27 | 59.53 ± 3.50 |
Both C. gladiata and C. ensiformis have a diploid chromosome number 2n = 22. The mitotic metaphase chromosomes are rather small. The TCL for C. gladiata and C. ensiformis are 40.46 ± 1.03 μm and 34.06 ± 3.87 μm, respectively. The individual metaphase chromosomes ranged from 4.72-2.63 μm long in C. gladiata, and from 4.21-2.43 μm long in C. ensiformis.
Both species have karyotypes composed of metacentric (m) chromosomes only (Table
Mitotic chromosomes (except for d, e, m, n) and interphase nuclei (d, e, m and n) of Canavalia gladiata (a–i, s, t) and Canavalia ensiformis (j–r, u, v) after sequential CPD staining and in situ hybridization. a, d, j, m CPD-stained chromosomes and interphase nuclei. c, e, l, n, s, u Chromosomes and interphase nuclei showing 5S (red) and 45S (green) rDNA signals produced by digoxigenin-labeled 5S rDNA and biotin-labeled 45S rDNA probes. b and k 5S and 45S rDNA signals only. f and o Signals produced by digoxigenin-labeled total genomic DNA of their own, g and p Chromosomes with sGISH signals. h and q Signals produced by digoxigenin-labeled total genomic DNA probes from other species. i and r Chromosomes with cGISH signals. t and vFISH of digoxigenin-labeled 5S rDNA repeats cloned from C. gladiata and C. ensiformis to same spreads shown in s and u, respectively. Arrows in a and j indicate positions of pair 7 centromeres. Arrowheads in a, i, j, s and u indicate distinguishable secondary constrictions (SC). Chromosome numbers in g and p are designated by karyotyping. Chromosomes in upper right corner of l are pair 6 from another spread showing proximal 5S rDNA loci on short arms. Chromosomes were counterstained using DAPI (blue). Bars = 10 µm.
CPD staining revealed that both species had similar fluorochrome banding patterns. The centromeric regions of all chromosome pairs and the 45S rDNA sites demonstrated by sequential rDNA-FISH appeared as red CPD bands (Fig.
FISH analyses of the 5S and 45S rDNA probes to the CPD-stained mitotic chromosomes and interphase nuclei are presented in Fig.
FISH performed on mitotic chromosomes using the cloned major 5S rDNA fragment probe generated signals in the regions corresponding to the 5S signals from pTa794 and in the centromeres wherein no signal was generated using pTa794 (Fig.
The chromosomal distribution patterns of repetitive DNA sequences were investigated using self-GISH. Distinct sGISH signal patterns were generated in both species and they were largely similar to each other (Figs
Idiograms of Canavalia gladiata (a, b) and C. ensiformis (c, d). a and c are idiograms displaying chromosome measurements and position and size of fluorochrome bands and rDNA FISH signals, b and d are idiograms displaying chromosome measurements and size and distribution of sGISH signals. Ordinate scale on left indicates relative chromosome length (% of haploid complement). The numbers above panel a are chromosome numbers.
cGISH was employed to probe the gDNA signals on the metaphase chromosomes of another species (Fig.
In this study, detailed karyotypes of C. gladiata and C. ensiformis were established using a combination of chromosome measurements, CPD bands, rDNA-FISH signals, and sGISH signals. The karyotypes provided the first molecular cytogenetic characterization of the two cultivated Canavalia species. The sGISH and rDNA-FISH signals were effective cytogenetic markers enabling unambiguous identificaion of individual chromosomes in both species.
The data revealed that the karyotypes of both C. gladiata and C. ensiformis are quite symmetrical. The karyotype of C. ensiformis has not been reported previously. The karyotype of C. gladiata in the present study shows more symmetry and differs from those described by
The rDNA-FISH revealed that there are a substantial number of 5S rDNA loci located in the centromeres in both species. There should be 5S rDNA repeats in all centromeres in both species because FISH using the cloned major 5S rDNA fragment generated weak signals in the centromeres wherein no signal was detected by pTa794. The copy number of 5S rDNA repeats within the centromeres of pair 7 (both species) and pair 3 of C. ensiformis was probably too low to be detected by FISH using the exogenous 5S rDNA probe. Centromeric 5S rDNA arrays have seldom been detected in plants by FISH. One to several centromeric 5S loci have only been reported for two Grindelia (Willdenow, 1807) species (
Another prominent feature of the two Canavalia genomes was the non-rDNA GC-rich heterochromatin in all centromeres (highlighted by CPD staining) (
The sGISH experiments revealed a distinct distribution of repetitive DNA sequences on the chromosomes of the two Canavalia species. sGISH data obtained from many plants showed that the chromosomal distribution of repetitive sequences is often non-uniform and forms clusters within heterochromatin blocks, and two different sGISH patterns may occur depending on the genome size of the species (
The molecular cytogenetic data obtained in this study revealed a high degree of similarity in genome organization between the two Canavalia species. This result confirms the evolutionary closeness between C. gladiata and C. ensiformis which was previously inferred from morphological and seed protein comparisons (
The data also revealed distinct differences between the two genomes. The genome size of C. ensiformis was nearly one-sixth less than that of C. gladiata based on their TCL (
Compared to C. gladiata, C. ensiformis gained an extra proximal 45S rDNA locus and a non-centromeric 5S rDNA locus but lost a centromeric 5S rDNA locus. Based on the signal intensity (
sGISH revealed that the distribution of repetitive sequences on pairs 5, 8, and 10, differed significantly between the two species. This fact suggests that C. ensiformis lost repetitive DNAs in some chromosomal regions and/or its chromosomes were rearranged during its evolution. Sequence analysis of 5S rDNA repeats revealed a lower level of NTS sequence identity between the species, indicating that their genomic sequences were clearly differentiated (
Individual chromosomes of both C. gladiata and C. ensiformis can be accurately identified by sGISH and rDNA-FISH signals.
Both C. gladiata and C. ensiformis genomes have particular characteristics including existence of non-rDNA GC-rich heterochromatin at all centromeres and 5S rDNA loci at the vast majority of centromeres, and a unique chromosomal distribution of repetitive DNA sequences.
Molecular cytogenetic comparison revealed both basic similarities and distinct differences in genome organization between C. gladiata and C. ensiformis, providing insights into the evolutionary relationships between them.
This work was supported by the Science and Technology Program of Hunan Province, China (No. 2013FJ4324) and the Natural Science Foundation of Hunan Province, China (No. 09JJ3063).