Research Article |
Corresponding author: Chao-Wen She ( shechaowen@aliyun.com ) Academic editor: Ekaterina Badaeva
© 2020 Chao-Wen She, Ying Mao, 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, Mao Y, Jiang X-H, He C-P (2020) Comparative molecular cytogenetic characterization of five wild Vigna species (Fabaceae). Comparative Cytogenetics 14(2): 243-264. https://doi.org/10.3897/CompCytogen.v14i2.51154
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To extend our knowledge on karyotype variation of the genus Vigna Savi, 1824, the chromosomal organization of rRNA genes and fluorochrome banding patterns of five wild Vigna species were studied. Sequential combined PI (propidium iodide) and DAPI (4',6-diamidino-2-phenylindole) (CPD) staining and fluorescence in situ hybridization (FISH) with 5S and 45S rDNA probes were used to analyze the karyotypes of V. luteola (Jacquin, 1771) Bentham, 1959, V. vexillata (Linnaeus, 1753) A. Richard, 1845, V. minima (Roxburgh, 1832) Ohwi & H. Ohashi, 1969, V. trilobata (Linnaeus, 1753) Verdcourt, 1968, and V. caracalla (Linnaeus, 1753) Verdcourt,1970. For further phylogenetic analysis, genomic in situ hybridization (GISH) with the genomic DNA of V. umbellata (Thunberg, 1794) Ohwi & H.Ohashi, 1969 onto the chromosomes of five wild Vigna species was also performed. Detailed karyotypes were established for the first time using chromosome measurements, fluorochrome bands, and rDNA-FISH signals. All species had chromosome number 2n = 2x = 22, and symmetrical karyotypes that composed of only metacentric or metacentric and submetacentric chromosomes. CPD staining revealed all 45S rDNA sites in the five species analyzed, (peri)centromeric GC-rich heterochromatin in V. luteola, V. trilobata and V. caracalla, interstitial GC-rich and pericentromeric AT-rich heterochromatin in V. caracalla. rDNA-FISH revealed two 5S loci in V. caracalla and one 5S locus in the other four species; one 45S locus in V. luteola and V. caracalla, two 45S loci in V. vexillata and V. trilobata, and five 45S loci in V. minima. The karyotypes of the studied species could be clearly distinguished by the karyotypic parameters, and the patterns of the fluorochrome bands and the rDNA sites, which revealed high interspecific variation among the five species. The V. umbellata genomic DNA probe produced weak signals in all proximal regions of V. luteola and all (peri)centromeric regions of V. trilobata. The combined data demonstrate that distinct genome differentiation has occurred among the five species during evolution. The phylogenetic relationships between the five wild species and related cultivated species of Vigna are discussed based on our present and previous molecular cytogenetic data.
Vigna species, karyotype, fluorochrome banding, fluorescence in situ hybridization (FISH), ribosomal RNA gene (rDNA)
The genus Vigna Savi, 1824, belonging to the tribe Phaseoleae of the family Fabaceae, includes over 100 species distributed throughout the Old and New Worlds (
The chromosomes of Vigna species were rather small in size and poorly morphologically differentiated (
FISH mapping of repetitive DNA sequences such as 5S and 45S rDNAs can not only generate useful landmarks for chromosome identification but can also provide valuable information on the evolutionary relationships between related species (e.g.
Detailed karyotypes can be constructed using the dataset of rDNA-FISH signals, fluorochrome bands and chromosome measurements, which reveals the genome organization of a plant species at chromosome level and is valuable in investigating the evolutionary relationships between related species (e.g.
In the present study, molecular cytogenetic characterization of five wild Vigna species, V. luteola, V. vexillata, V. minima, V. trilobata and V. caracalla was conducted using sequential CPD staining and dual color FISH with 5S and 45S rDNA probes. Detailed karyotypes of the five species were established using a combination of chromosome measurements, fluorochrome bands, and rDNA-FISH signals. Six different parameters of karyotype asymmetry were calculated for the elucidation of karyotype variation among these species. cGISH with V. umbellata genomic DNA probe onto the somatic chromosomes of the five species, the method that was applied in the molecular-cytogenetic study on the seven cultivated Vigna species (
Seeds of V. luteola (Jacquin, 1771) Bentham, 1959 (PI 406329), V. vexillata (Linnaeus, 1753) A.Richard, 1845 (PI 406428, Origin traced to PI 225934), V. minima (Roxburgh, 1832) Ohwi & H. Ohashi, 1969 (PI 483081), V. trilobata (Linnaeus, 1753) Verdcourt, 1968 (PI 286306), V. caracalla (Linnaeus, 1753) Verdcourt, 1970 (Synonym of Cochliasanthus caracalla (Linnaeus, 1753) Trew, 1764; PI 146800), and V. umbellata (Thunberg, 1794) Ohwi & H. Ohashi, 1969 (PI 208460) were obtained from the U.S. National Plant Germplasm System. Genomic DNA of V. umbellata was isolated from young leaves using Rapid Plant Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China).
Mitotic metaphase chromosome spreads were prepared as previously described with minor modification (
CPD staining followed the procedure described by
A 45S rDNA clone containing a 9.04-kb tomato 45S rDNA insert (
FISH with the 5S and 45S rDNA probes, and cGISH with V. umbellata genomic DNA probe were performed after CPD staining on the same slides. The slides previously stained by CPD were washed in 2× SSC, twice for 15 min each, dehydrated through an ethanol series (70%, 90%, and 100%, 5 min each) and then used for hybridization. The in situ hybridization methodology followed the protocol described by
The karyotyping methodology followed that described by
Representative mitotic chromosomes of the five species studied are shown in Figure
Mitotic chromosomes from V. luteola (A, B), V. vexillata (C, D), V. minima (E, F), V. trilobata (G, H), and V. caracalla (I–L) stained using CPD method and sequential dual-colour FISH with digoxigenin-labelled 5S and biotin-labelled 45S rDNA probes. A, C, E, G, I are the chromosomes stained using CPD. The chromosome numbers are designated by karyotyping. B, D, F, H, J are the chromosomes displaying the 5S (red) and 45S rDNA (green) signals. The total DNA was counterstained using DAPI (blue). K, L are DAPI and PI grey scale images of the V. caracalla chromosomes stained using CPD, respectively. The images are converted to reverse images with Photoshop software. Arrows and arrowheads in I indicate the satellites and interstitial CPD bands, respectively. Scale bars: 10 µm.
Karyotypic parameters of the five wild Vigna species (all, 2n = 2x = 22).
Species | KF | TCL ± SE (μm) | C (μm) | RRL | CI±SE | A1 | A2 | As K (%) | AI | Stebinns’ types |
V. luteola | 11m | 33.81 ± 1.56 | 3.07 | 6.88–12.40 | 44.35 ± 2.45 | 0.20 | 0.21 | 55.97 | 1.15 | 1A |
V. vexillata | 11m | 25.67 ± 2.02 | 2.33 | 6.99–12.66 | 43.24 ± 3.45 | 0.23 | 0.19 | 57.01 | 1.52 | 1A |
V. minima | 11m | 38.29 ± 1.04 | 3.48 | 7.37–12.14 | 44.55 ± 2.03 | 0.19 | 0.14 | 55.53 | 0.66 | 1A |
V. trilobata | 9m + 2sm | 36.56 ± 2.73 | 3.32 | 7.20–13.48 | 42.15 ± 3.87 | 0.27 | 0.19 | 58.00 | 1.76 | 1A |
V. caracalla | 10m (1SAT) + 1sm | 46.62 ± 1.71 | 4.24 | 5.61–12.80 | 44.37 ± 3.13 | 0.20 | 0.20 | 55.39 | 1.41 | 1B |
All the five Vigna species studied have diploid chromosome number 2n = 2x = 22. The metaphase chromosomes were small, with a mean chromosome length between 2.33 μm (V. vexillata) and 4.24 μm (V. caracalla). The total length of the haploid complement (TCL) ranged from 25.67 μm to 46.62 μm, and the mean centromeric index (CI) of the complements varied between 42.15 ± 3.87 (V. trilobata) and 44.55 ± 2.03 (V. minima). V. caracalla exhibited the most variation in chromosome length, and V. trilobata was characterized by the highest level of variation in the centromeric index.
The karyotypes of V. luteola, V. vexillata, V. minima were composed of metacentric (m) chromosomes only, while those of V. trilobata and V. caracalla were composed of metacentric and submetacentric (sm) chromosomes (Table
Idiograms of the five Vigna species that display the chromosome measurements, and the position and size of the fluorochrome bands and rDNA-FISH signals. A–E indicate V. luteola, V. vexillata, V. minima, V. trilobata, and V. caracalla, 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 chromosomes 1 to 11.
CPD staining revealed distinct heterochromatin differentiation among the five species studied (Figs
GISH with Vigna umbellata genomic DNA probe (red) to the chromosomes of V. luteola (A, B), V. vexillata (C), V. minima (D, E), V. trilobata (F, G), and V. caracalla (H, I). A, D, F, H CPD banded chromosomes before the hybridization procedure. The chromosomes shown in C is the same spread shown in Figure
FISH results of the 5S and 45S rDNA probes to the CPD-stained mitotic chromosomes are presented in Figure
The FISH patterns of 5S and 45S rDNAs of the five species displayed conspicuous interspecific variation. Among the five taxa, V. luteola, V. vexillata, V. minima and V. trilobata had a single pair of 5S rDNA sites, while V. caracalla had two pairs of 5S sites (Figs
For the 45S rDNA sites, there was considerable variation in number, size and position among the five taxa analyzed (Table
The distribution of fluorochrome bands and rDNA sites in the five wild Vigna species.
Species | Fluorochrome bands | Number (pairs) and location of rDNA sites† | ||||
Type | Distribution† | Amount (%)‡ | Band size (mean)§ | 5S| | 45S| | |
V. luteola | CPD | all CENs, PCENs and 45S sites | 29.19 | 1.98–3.21 (2.65) | one [11S-PCEN (16.55%)] | one (3S) |
V. vexillata | CPD | all 45S sites | one [(8L-INT(52.29%)] | two [2S-TER (20.53%), 3S-TER (16.73%)] | ||
V. minima | CPD | all 45S and 5S sites | one [2S-INT (30.86%)] | five [2L-TER (58.64%), 4L-TER (59.42%), 6S-TER (38.91%), 7S-TER (50.74%), 9S-TER (67.94%)] | ||
V. trilobata | CPD | all CENs, PCENs and 45S sites | 20.04 | 2.73–1.12 (1.82) | one [4L-PCEN(14.95%)] | two [6S-PCEN(25.05%), 7S] |
V. caracalla | CPD | all CENs, PCENs and 45S sites, 4S-, 4L-, 5L-INTs | 21.68¶ | 0.89–2.63 (1.55) | two [2L-INT(34.32%), 5S-INT (56.7%)] | one (1S) |
DAPI | 2, 3, 4, 5S-PCENs; 4, 5, 6, 8L-PCENs | 8.19 | 0.69–1.38 (1.04) |
Comparative genomic in situ hybridization with V. umbellata genomic DNA probe was employed to reveal the homology of repetitive DNA sequences between V. umbellata and the five wild Vigna species (Fig.
In the current study, detailed karyotypes of V. luteola, V. vexillata, V. minima, V. trilobata and V. caracalla are established using a dataset of chromosome measurements, fluorochrome bands, and rDNA-FISH signals, thus providing the first primary molecular cytogenetic characterization of these wild Vigna species. Although FISH mapping of rDNAs in V. vexillata var. tsusimensis Matsumura, 1902 has been conducted (Chio et al. 2013), but the detailed karyotype of this species has not yet been established. Our results reveal that the karyotypic parameters and patterns of the fluorochrome bands and rDNA sites vary among the five Vigna species studied, enabling an accurate distinguishment between individual genomes.
This study identifies the chromosome number of all the five species as 2n = 22, in accordance with that reported previously by other authors (
The results reveal significant variation in karyotype length (TCL) among the five taxa studied. For example, the TCL of V. caracalla was 1.82 times longer than that of V. vexillata. Except V. caracalla, the TCLs of the other four wild species were much shorter than those of the seven cultivated Vigna species obtained previously by us (
The significant variation in CPD and DAPI+ bands, with regard to appearance, position and size, reflects distinct GC-rich and AT-rich heterochromatin differentiation among the five wild Vigna species (
With the exception of the rDNA CPD bands, V. luteola, V. trilobata, and V. caracalla also displayed centromeric and pericentromeric non-rDNA CPD bands. Especially, V. caracalla possessed interstitial non-rDNA CPD bands, which have not been observed in other Vigna species (
The occurrence of the pericentromeric DAPI+ bands in V. caracalla was another conspicuous heterochromatic differentiation of this species. Among the Vigna species previously analyzed by fluorochrome banding technique, AT-rich heterochromatin blocks have been observed in the pericentromeric regions of several chromosome pairs of V. radiata (
To date, FISH mapping of rDNA sites has been reported only for V. vexillata var. tsusimensis among the wild species within the genus Vigna (
Our rDNA-FISH results reveal considerable variations in number, position and even size of both 45S and 5S rDNA sites among the five wild Vigna species studied. Similarly, wide interspecific differences in the pattern of rDNA sites were observed among the seven cultivated Vigna species (
In the early time, the Vigna genus was divided into seven subgenera (
Among the remaining four wild Vigna species analyzed, both V. luteola and V. vexillata are of African origin being categorized into Vigna subg. Vigna and subg. Haydonia, respectively (
Molecular cytogenetic karyotypes of five wild Vigna species, V. luteola, V. vexillata, V. minima, V. trilobata and V. caracalla are established for the first time using fluorochrome banding and rDNA-FISH techniques. Comparative molecular cytogenetic karyotyping reveals distinct variations in the karyotypic parameters, and the patterns of the fluorochrome bands and rDNA sites among species, enabling an accurate distinguishment between individual genomes. The molecular cytogenetic data of the five species is helpful to clarify the phylogenetic relationships among related Vigna species.
This work was supported by the Natural Science Foundation of Hunan Province, China (No. 09JJ3063 and No. 2019JJ40231) and the Foundation of Hunan Double First-rate Discipline Construction Projects, China.
Table S1. Chromosome measurements of the five wild Vigna species obtained from five metaphases per species
Data type: species data