Research Article |
Corresponding author: Chao-Wen She ( shechaowen@aliyun.com ) Academic editor: Lorenzo Peruzzi
© 2024 Yan-Fang Wei, Xiang-Hui Jiang, Rong Song, Chao-Wen She.
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:
Wei Y-F, Jiang X-H, Song R, She C-W (2024) Molecular cytogenetic characterization of 9 populations of four species in the genus Polygonatum (Asparagaceae). Comparative Cytogenetics 18: 73-95. https://doi.org/10.3897/compcytogen.18.122399
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To characterize the chromosomes of the four species of Polygonatum Miller, 1754, used in traditional Chinese medicine, P. cyrtonema Hua, 1892, P. kingianum Collett et Hemsley, 1890, P. odoratum (Miller, 1768) Druce, 1906, and P. sibiricum Redouté, 1811, and have an insight into the karyotype variation of the genus Polygonatum, fluorescence in situ hybridization (FISH) with 5S and 45S rDNA oligonucleotide probes was applied to analyze the karyotypes of 9 populations of the four species. Detailed molecular cytogenetic karyotypes of the 9 populations were established for the first time using the dataset of chromosome measurements and FISH signals of 5S and 45S rDNA. Four karyotype asymmetry indices, CVCI, CVCL, MCA and Stebbins’ category, were measured to elucidate the asymmetry of the karyotypes and karyological relationships among species. Comparison of their karyotypes revealed distinct variations in the karyotypic parameters and rDNA patterns among and within species. The basic chromosome numbers detected were x = 9, 11 and 13 for P. cyrtonema, x = 15 for P. kingianum, x = 10 and 11 for P. odoratum, and x = 12 for P. sibiricum. The original basic chromosome numbers of the four species were inferred on the basis of the data of this study and previous reports. All the 9 karyotypes were of moderate asymmetry and composed of metacentric, submetacentric and subtelocentric chromosomes or consisted of two of these types of chromosomes. Seven populations have one locus of 5S rDNA and two loci of 45S rDNA, and two populations added one 5S or 45S locus. The karyological relationships among the four species revealed by comparison of rDNA patterns and PCoA based on x, 2n, TCL, CVCI, MCA and CVCL were basically accordant with the phylogenetic relationships revealed by molecular phylogenetic studies. The mechanisms of both intra- and inter-specific dysploidy in Polygonatum were discussed based on the data of this study and literature.
Cytotaxonomy, fluorescence in situ hybridization, karyotype, karyotype asymmetry, Polygonatum, ribosomal RNA genes (rDNA)
The genus Polygonatum Miller, 1754, as the largest genus in the tribe Polygonateae (Asparagaceae), comprises ca. 70 species (
Polygonatum species show a high variation in morphology and a wide overlap in geographical distribution, which makes infrageneric classification and species identification very complicated (
The classification of Polygonatum has long been controversial.
The ribosomal genes, 45S (18S-5.8S-26S) and 5S rDNAs, are organized in tandem arrays with high copy numbers, and then widely utilized as probes for FISH in plants. The rDNA FISH signals can be used as informative markers for a better characterization of the chromosomes of plant species, revealing genome organization at molecular cytogenetic level (e.g.
In the present study, comparative molecular cytogenetic analysis of 9 populations of four Polygonatum species, P. cyrtonema, P. kingianum, P. sibiricum and P. odoratum, was conducted using dual-color FISH with 5S and 45S rDNA oligonucleotide probes. Detailed molecular cytogenetic karyotypes of these populations were quantitatively established using a combination of chromosome measurements and rDNA FISH signals. Four different karyotype asymmetry indices of each population were calculated for evaluating asymmetry of the karyotypes and karyological relationships among the populations. The combined data of karyotypic parameters and rDNA patterns were assessed to gain insights into the intra- and inter-specific karyotype differentiation as well as the phylogenetic relationships among the four species.
Plants of 9 populations including four of P. cyrtonema, two of P. kingianum, two of P. odoratum and one of P. sibiricum (Suppl. material
The rhizomes used for cytogenetic experiments were cultivated in pots with mixed planting soil consisting of humus soil and sandy soil, and young new roots grew from the rhizomes in about 10 to 14 days. Chromosome spreads were prepared using a protocol previously published by us (
The 5S rDNA oligonucleotide probes 5S-1 and 5S-2 and the 45S rDNA oligonucleotide probes 45S-1, 45S-2 and 45S-3, which were described previously by
FISH was performed according to the procedure described by
The slides were washed in 2 × SSC twice each for 5 min at room temperature after hybridization. Then, the chromosomes were counterstained with 3 µg ml−1 DAPI in 30% (v/v) Vectashield H-1000 and visualized with an Olympus BX60 microscope equipped with a QImaging Retiga R6 CCD camera (Teledyne Photometrics, Canada) which was controlled using Ocular software (Teledyne Photometrics, Canada). Observations were made using UV, blue and green excitation filters for DAPI, 6-FAM, and TAMRA, respectively. Grey-scale images were digitally captured and merged by the Ocular software. The final images were adjusted with Adobe Photoshop CS 8.01.
The methodology of karyotype analysis described recently by us was used (
Bidimensional scatter diagram for the 9 populations with MCA vs. CVCL was plotted in order to visualize karyotype asymmetry relationships among them. To determine the karyological relationships among the 9 populations, a principal coordinate analysis (PCoA) using Gower’s similarity coefficient were performed based on six quantitative parameters (x, 2n, TCL, CVCI, MCA and CVCL) according to the proposal by
The general karyotype features of the 9 populations of P. cyrtonema, P. kingianum, P. odoratum and P. sibiricum are listed in Table
FISH to metaphase chromosomes of 9 populations of four Polygonatum species, P. cyrtonema (Pc), P. kingianum (Pk), P. odoratum (Po) and P. sibiricum (Ps), using 5S rDNA (green) and 45S rDNA (red) oligonucleotide probes. Chromosomes were counterstained with DAPI (blue). The chromosome numbers were designated by karyotyping A Pc AHDBS B Pc HNHH C Pc HBHS D Pc SCSN E Pk YNKM F Pk YNWS G Po HNXH H Po AHDBS I Ps HNFNS. Scale bars: 10 µm.
Idiograms of 9 populations of four Polygonatum species, P. cyrtonema (Pc), P. kingianum (Pk), P. odoratum (Po) and P. sibiricum (Ps), that display the chromosome measurements, and the location and size of the 5S (green) and 45S (red) rDNA FISH signals A Pc AHDBS B Pc HNHH C Pc HBHS D Pc SCSN E Pk YNKM F Pk YNWS G Po HNXH H Po AHDBS I Ps HNFNS. The ordinate scale on the left indicates the relative length of the chromosomes (i.e. % of haploid complement). The numbers at the bottom indicate the the serial number of chromosomes.
Populations* | Karyotype formula (KF) | TCL ± SE (μm) | C (μm) | RRL | CI ± SE | CV CI | CV CL | MCA | Stebinns’ types |
---|---|---|---|---|---|---|---|---|---|
Pc AHDBS | 2n = 18 = 10m(2SAT) + 6sm + 2st(2SAT) | 72.23 ± 8.83 | 8.03 | 6.23–17.01 | 33.45 ± 7.10 | 21.99 | 34.38 | 31.46 | 2B |
Pc HNHH | 2n = 22 = 4m + 16sm(4SAT) + 2st | 80.34 ± 20.91 | 7.30 | 5.46–11.95 | 31.84 ± 7.33 | 23.02 | 34.85 | 36.28 | 3B |
Pc HBHS | 2n = 22 = 6m + 10sm(2SAT) + 6st(2SAT) | 72.39 ± 5.03 | 6.58 | 5.01–13.08 | 32.46 ± 8.32 | 24.59 | 38.51 | 35.07 | 3B |
Pc SCSN | 2n = 26 = 8m + 14sm(4SAT) + 4st | 84.07 ± 6.93 | 6.47 | 5.21–10.41 | 32.22 ± 6.25 | 19.40 | 22.94 | 35.56 | 3A |
Pk YNKM | 2n = 30 = 6m + 10sm(2SAT) + 14st(4SAT) | 93.40 ± 12.14 | 6.23 | 2.85–10.13 | 28.05 ± 9.51 | 33.92 | 43.90 | 43.90 | 3B |
Pk YNWS | 2n = 30 = 10m + 8sm(2SAT) + 12st(2SAT) | 80.38 ± 6.61 | 5.36 | 3.34–10.61 | 31.26 ± 10.62 | 33.98 | 42.16 | 37.48 | 3B |
Po HNXH | 2n = 20 = 8m(2SAT) + 12sm(2SAT) | 86.32 ± 8.18 | 8.63 | 5.95–11.61 | 33.86 ± 7.79 | 23.00 | 31.47 | 32.28 | 2A |
Po AHDBS | 2n = 22 = 6m + 10sm(2SAT) + 6st(2SAT) | 88.61 ± 10.32 | 8.01 | 4.90–12.82 | 30.90 ± 8.85 | 28.65 | 36.88 | 38.20 | 3B |
Ps HNFNS | 2n = 24 = 12sm(2SAT) + 12st(2SAT) | 76.14 ± 5.30 | 6.35 | 5.91–11.16 | 28.30 ± 5.85 | 20.67 | 23.61 | 43.40 | 3A |
The four populations of P. cyrtonema have three different chromosome numbers: 2n = 18 for Pc AHDBS, 2n = 22 for Pc HNHH and Pc HBHS, and 2n = 26 for Pc SCSN, among which 2n = 26 is reported for the first time. Both populations of P. kingianum have the same chromosome number 2n = 30. The chromosome numbers of the two populations of P. odoratum are different: 2n = 20 for Po HNXH, 2n = 22 for Po AHDBS. The chromosome number of P. sibiricum is 2n = 24. Among the 9 populations, the total length of the haploid complement (TCL) ranges from 72.23 μm (Pc AHDBS) μm to 93.40 μm (Pk YNKM) with a mean chromosome length between 5.36 μm (Pk YNWS) and 8.63 μm (Po HNXH), showing both inter- and intra-specific variation. According to the classification of
The karyotypes are composed of m, sm and st chromosomes or consisted of two of these types of chromosomes (Table
The values of the four karyotype asymmetry indices including CVCI, CVCL, MCA and Stebbins’ type are presented in Table
The karyotype asymmetry relationships among the 9 populations that are expressed by means of bidimensional scatter plot of MCA vs. CVCL are illustrated in Fig.
Bidimensional scatter plot of MCA vs. CVCL for the 9 populations of four Polygonatum species, P. cyrtonema (Pc), P. kingianum (Pk), P. odoratum (Po) and P. sibiricum (Ps).
PCoA based on the six quantitative karyological parameters reveals the karyological relationships among the 9 populations (Fig.
PCoA for the 9 populations of four Polygonatum species, P. cyrtonema (Pc), P. kingianum (Pk), P. odoratum (Po) and P. sibiricum (Ps), based on x, 2n, TCL, MCA, CVCL and CVCI. Pc1, Pc2, Pc3 and Pc4 represent Pc AHDBS, Pc HNHH, Pc HBHS and Pc SCSN, respectively. Pk1 and Pk2 represent Pk YNKM and Pk YNWS, respectively. Po1 and Po2 represent Po HNXH and Po AHDBS, respectively. Ps represents Ps HNFNS. PCoA1 reflects the original data characteristics before the dimensionality reduction of 57.84%. PCoA2 reflects the character of the original data before the dimensionality reduction of 25.78%. The sum of the two percentages is 83.62%, indicating that the two-dimensional coordinate system can reflect the characteristics of 83.62% of the original data.
The FISH results show inter- and intra-specific variations in number and location of 5S and 45S rDNA loci (Figs
The number (pair) and location of rDNA loci in 9 populations of four Polygonatum species.
Populations† | 5S rDNA‡ | 45S rDNA‡ |
---|---|---|
Pc AHDBS | one[8L-DIS(78.73%)] | two[3L-INT§(57.57%),6L-INT§(65.28%)] |
Pc HNHH | one[10L-INT(61.65%)] | two[1L-INT(49.19%)§,4L-INT(57.46%)§] |
Pc HBHS | two[2L-PRO(19.28%),10L-DIS(79.08%)] | two[1L-INT(45.14%)§,4L-INT(59.20%)§] |
Pc SCSN | one[9L-DIS(78.08%)] | three[3S-INT(44.35%)§, 8S-DIS(82.02%)§, 10L-INT(46.75%)] |
Pk YNKM | one[1S-PRO(23.36%)] | three[4L-INT(61.57%)§,7L-INT(65.24%)§, 8L-INT(70.65%)§] |
Pk YNWS | one[2S-PRO(21.29%)] | two[3L-INT(44.50%)§, 8L-INT(70.88%)§] |
Po HNXH | one[9L-INT(58.06%)] | two[2L-INT(57.05%)§, 5L-INT(65.56%)§] |
Po AHDBS | one and a half [1L-PRO(18.29%)| , 10L-DIS(82.99%)] | two[2L-INT(65.41%)§, 5L-INT(54.92%)§] |
Ps HNFNS | one[11L-INT(71.79%)] | two[1S-INT(33.76%)§, 3L-INT(71.86%)§] |
As for 45S rDNA site, there exist two loci in Pc AHDBS, Pc HNHH, Pc HBHS, Pk YNWS, Po HNXH, Po AHDBS and Ps HNFNS and three loci in Pc SCSN and Pk YNKM (Figs
In the present study, a primary molecular cytogenetic characterization of 9 populations of P. cyrtonema, P. kingianum, P. odoratum and P. sibiricum is conducted for the first time. The karyotypic parameters and rDNA patterns vary among the populations studied, enabling an accurate distinguishment between individual genomes. The rDNA FISH signals provide new chromosomal markers for investigating the inter- and intra-specific karyotype evolution in the genus Polygonatum.
The evolution of chromosome number in Polygonatum is mainly dysploidy, and a few species have polyploidy (
The scatter plot of MCA vs. CVCL reveals that the karyotypic structures vary both among species and among different populations of the same species in terms of both intra- and inter-chromosomal asymmetry (Fig.
Chromosome arrangements also occur between populations with the same basic chromosome number. The karyotypes of Pc HNHH and Pc HBHS (both x = 11) show some differences, mainly including significant changes of the arm ratios of pairs 2 and 8 between the two populations, and the occurrence of another 5S locus on pair 2 in Pc HBHS (Fig.
Although the chromosomal rearrangements inferred from the changes in chromosomal morphology and rDNA pattern may only represent the tip of the iceberg of the dysploidy within species of the genus Polygonatum. However, it has been revealed that, in the evolutionary process, geographically diverse populations of Polygonatum species are easy to preserve large-scale and multiple chromosomal rearrangements. The reasons for this may be the perennial and clonal nature of Polygonatum species (
The direction of the basic chromosome number evolution in the interspecific dysploidy of Polygonatum has long been an important and challenging issue in the cytogenetic study of the genus. From our comparative molecular cytogenetic karyotype analysis, there are obvious differences in chromosome number, karyotypic structure and rDNA pattern among P. cyrtonema and P. odoratum (representatives of sect. Polygonatum), P. kingianum (a representative of sect. Verticillata) and P. sibiricum (the representative of sect. Sibirica) (
According to the infrageneric classification system of
The similarities and differences in rDNA patterns among species reflect the closeness of relatedness between species (e.g.
PCoA based on x, 2n, TCL, CVCI, MCA, CVCL is – thus far – the most legitimate approach to use for comparing karyotypes and reconstructing karyological relationships among taxa (
Detailed molecular cytogenetic karyotypes of 9 populations of four Polygonatum species, P. cyrtonema, P. kingianum, P. odoratum and P. sibiricum, are established for the first time using the dataset of chromosome measurements and FISH signals of 5S and 45S rDNA. Comparative karyotyping reveals distinct variations in the karyotypic parameters and rDNA patterns among and within species, and intraspecific dysploidy of P. cyrtonema and P. odoratum. The evolutionary relationships among the four species revealed by rDNA pattern comparison and PCoA based on x, 2n, TCL, CVCI, MCA and CVCL are basically accordant with the phylogenetic relationships revealed by molecular phylogenetic studies.
This work was supported by the Natural Science Fundation of Hunan Province, China (2019JJ40231).
Chao-Wen She https://orcid.org/0000-0003-1935-5509
The plant materials
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Chromosome measurements of 9 populations of four Polygonatum species
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Metaphase chromosomes of 9 populations of four Polygonatum species, P. cyrtonema (Pc), P. kingianum (Pk), P. odoratum (Po) and P. sibiricum (Ps)
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Diagrams of the distribution of basic chromosome numbers within four Polygonatum species, P. cyrtonema, P. kingianum, P. odoratum and P. sibiricum
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