Twenty-four Miscanthus accessions comprised of 6 M. sinensis, 6 M. floridulus, 6 M. sacchariflorus and 6 M. lutarioriparius were selected from different provinces of China and planted in the Miscanthus germplasm nursery located at the Hunan Agricultural University (Table 1). All the materials were used for ITS sequence analysis. Meanwhile, for CPD staining, rDNA FISH, karyotype analysis and cGISH, No. 03 (M. sinensis), No. 10 (M. floridulus), No. 16 (M. sacchariflorus) and No. 21 (M. lutarioriparius) were used. Imperata cylindrica (Beauvois, 1812) was included as an outgroup for the ITS phylogenetic analysis, and its sequences (JN407505.1) were obtained from GenBank Database.

Total genomic DNA (gDNA) was extracted from fresh leaf tissue using the cetyltrimethylammonium bromide (CTAB) method described by Murray and Thompson (1980). The quality and concentration of DNA were measured by 1% agarose gel electrophoresis and a microplate reader (BioTek Instruments Inc, Winooski, USA).

Mitotic metaphase chromosomes were prepared by using the root tips according to the procedure described by She et al. (2006). The actively growing root tips were collected from potted plants and treated with saturated α-bromonaphthalene for 1.5 h at 28 °C, fixed in 3:1 (v/v) methanol/glacial acetic acid for at least 12 h at room temperature, and then stored at 4 °C until use. The fixed root tips were then washed in double distilled water and citrate buffer (0.01 mM citric acid-sodium citrate, pH 4.5) for 10 min each and incubated in a mixture of 2% cellulase R-10 (Yakult Pharmaceutical Industry, Tokyo, Japan), 2% pectolyase Y-23 (Yakult Pharmaceuticals), and 2% macerozyme R-10 (Sigma-Aldrich, Steinhem, Germany) in citric acid buffer at 28 °C for 2.5~3 h. Root tips were transferred to a glass slide along with the fixative and dissected using fine-pointed forceps. Finally, the slides were dried above a flame. Good preparations were selected by Olympus BX60 phase contrast microscope, and then stored at -20 °C.

The CPD staining followed the procedure described in She et al. (2006). Chromosome preparations were treated with RNase A and pepsin then stained with a mixture of 3 μg/ml DAPI and 0.6 μg/ml PI (both from Sigma-Aldrich) in a 30% (v/v, using double-distilled water as solvent) solution of Vectashield H-1000 (Vector Laboratories Burlingame, USA). Preparations were examined under an Olympus BX60 epifluorescence microscope equipped with a CoolSNAP EZ CCD camera (Photometrics, Tucson, USA). The CCD camera was controlled using Ocular software (Molecular Devices, Sunnyvale, USA). Photographs were taken using a green excitation filter for PI and a UV excitation filter for DAPI. DAPI and PI grey-scale images of the same plate were merged to produce a CPD image. The final images were optimized for contrast and background using Adobe PHOTOSHOP CS8.0.

A 45S rDNA clone containing a 9.04-kb tomato 45S rDNA insert (Perry and Palukaitis 1990) were used as probe to localize the 18S-5.8S-26S ribosomal RNA gene. The DNAs (45S rDNA and gDNAs) of the four Miscanthus species were labeled with biotin-16-dUTP and digoxigenin-11-dUTP, respectively, using the Nick Translation Kit (Roche Diagnostics, Mannheim, Germany).

FISH with the 45S rDNA probe was carried out on the same slides previously stained with CPD. FISH with the M. sinensis, M. floridulus, M. sacchariflorus and M. lutarioriparius genomic probes to the M. sinensis chromosomes. FISH with the M. sacchariflorus, M. floridulus and M. lutarioriparius genomic probes to the M. sacchariflorus chromosomes, and FISH with the M. lutarioriparius and M. floridulus genomic probes to the the M. lutarioriparius chromosomes were performed, respectively. The slides previously stained or hybridized were washed twice for 15 min each in 2 × SSC, dehydrated through an ethanol series (70%, 90% and 100%, 5 min each) and then used for hybridization. The in situ hybridization and detection were performed as described by She et al. (2006). Briefly, 40 μl of the hybridization mixture, which contained 20 μl 20% dextran sulfate, 1 μl ssDNA, 16 μl hybridization buffer (HB50, containing 50% deionized formamide and 50 mM sodium phosphate, pH 7.5) and 3 μl labeled DNA (final concentration 100–150 ng/slide), was added to each slide and covered with a 24 × 50 mm glass coverslip. Chromosomes and probe were denatured together on ThermoBrite S500-24 (Abbott Molecular, USA) at 80 °C for 3 min, and then were incubated at 37 °C for 24 h. Post-hybridization washing was performed in 0.1 × SSC two times for 15 min each at 42 °C, followed by rinsing in 2 × SSC three times for 5 min each at 42 °C and in TN buffer (containing 100 mM Tris-HCl and 150 mM NaCl, pH 7.5) for 5 min at room temperature. Hybridization signals were detected after incubating the slides with 100 μl TNB buffer (0.5% Roche blocking reagent in TN buffer) for 30 min at 37 °C, and followed by rinsing in TN buffer for 1 min at room temperature.

The biotin-labeled 45S rDNA was detected using Fluorescein Avidin D (Vector Laboratories). The digoxigenin-labeled gDNA was detected using Anti-digoxigenin-rhodamine (Roche Diagnostics). The specific steps were as follows: 100 μl of 1% Fluorescein Avidin D or Anti-digoxigenin-rhodamine, diluted with TNB buffer, was added to each slide and covered with a glass coverslip, and then were incubated at 37 °C in dark for 1 h. Afterwards, the coverslip was removed and rinsed with TN buffer three times for 5 min each in dark. Slides were counterstained with 3 µg/ml DAPI in a 30% solution of Vectashield H100 and subsequently examined under an epifluorescence microscope equipped with the CCD camera as mentioned above. Observations were made using a UV, blue and green excitation filters for DAPI, fluorescein, and rhodamine, respectively. Grey-scale images were digitally captured and merged by the Ocular software. The final images were adjusted with Adobe PHOTOSHOP CS8.0.

For each species, five well-spread metaphase plates were measured using Adobe PHOTOSHOP CS8.0 to obtain the chromosome relative lengths (RL; % of haploid complement), arm ratios (AR = long arm/short arm), chromosome length ratio (longest chromosome length / shortest chromosome length), size of the fluorochrome band (expressed as a percentage of the karyotype length) and the percentage distance from the rDNA site to the centromere (di = d × 100/a; d = distance of the centre of the rDNA sites from the centromere; a = length of the corresponding chromosome arm) (Greilhuber and Speta 1976). In addition, the total length of the haploid complement (TCL; i.e. karyotype length) was measured using the five metaphase cells with the highest degree of chromosome condensation. The arm ratio was used to classify the chromosomes according to the system described by Levan et al. (1964). Karyotype asymmetry was determined using the mean centromeric index (CI), the intrachromosomal asymmetry index (A1), the interchromosomal asymmetry index (A2) (Zarco 1986), the ratio of the length of all long arms in the chromosome set to the total chromosome length in set (As K%) (Arano 1963), the asymmetry index (AI) (Paszko 2006), and the categories of Stebbins (Stebbins 1971). The chromosomes were arranged in order of decreasing lengths. Idiograms were drawn based on chromosomes measurement data, fluorochrome bands and 45S rDNA FISH signals.

The rDNA-ITS regions (including ITS1, 5.8s and ITS2) of the four Miscanthus species were amplified using the universal primers ITS4 and ITS5 (ITS4 primer sequence: 5’-TCCTCCGCTTATTGATATGC-3’, ITS5 primer sequence: 5’-GGAAGTAAAAGTCGTAACAAGG-3’) (White et al. 1990). The total volume of the PCR amplification reaction was 25 μl, including 2.5 μl 10 × PCR buffer, 1.5 μl 25 mM MgCl₂, 0.5 μl 10 mM dNTP, 0.75 μl 10 μM of each primer, 0.5 μl Taq DNA polymerase (Sangon, Shanghai, China), 1.5 μl gDNA (30~50 ng/μl) and 17 μl ddH₂O. The amplification conditions were: pre-denatured at 95 °C for 4 min; denaturation at 94 °C for 45 s, annealing at 54 °C for 45 s, extension at 72 °C for 45 s, 38 cycles; and a final extension step of 10 min at 72 °C on a thermal cycler PTC-200. The PCR products were detected by 1% agarose gel electrophoresis. PCR products purification and ITS sequencing were performed by Sangon. The ITS sequences have been deposited in GenBank and the accession numbers are listed in Table 1.

Each DNA sequence was spliced by bi-directional sequencing. Then, the similarity searches were performed using the NCBI (National Centre for Biotechnology Information), BLAST network service. Sequences were aligned with CLUSTAL W program. The MEGA 7.0 software (Kumar et al. 2016) was used for sequence analyses (estimating percentage of the G + C content, variable sites and parsimony informative sites). ITS sequences of I. cylindrical was used as the outer group plant, and phylogenetic analyses were carried out using the neighbour joining (NJ) and maximum parsimony (MP) methods. In the ITS phylogenetic tree, the confidence of each branch was tested using bootstrap (Felsenstein 1985), each performing 1 000 cycles to evaluate the systematic significance and reliability of each branch.

The karyotype measurement data for the four Miscanthus species are listed in Suppl. material 1, Tables S1–S4. The general karyotype features and parameters for the four Miscanthus species are listed in Table 2. Representative mitotic chromosomes, karyotypes showing the fluorescent bands and ideograms are shown in Figs 1–3.

Figure 1.

Mitotic chromosomes from M. sinensis (A, B), M. sacchariflorus (C, D), M. lutarioriparius (E, F) M. floridulus (G, H), stained with CPD staining and sequentially FISH with biotin-labelled 45S rDNA probe. A, C, E and G are chromosomes stained using CPD. The chromosome numbers were designated by karyotyping B, D, F and H are the chromosomes showing the 45S (green) signals. Arrowheads in A and G indicate the blue DAPI bands. Scale bars: 10 μm.

Figure 2.

Karyotype showing CPD and DAPI bands of M. sinensis (A), M. floridulus (B), M. sacchariflorus (C), M. lutarioriparius (D). Arrowheads in A and B indicate the blue DAPI bands.

Figure 3.

Idiograms of the four Miscanthus species that display the chromosome measurements, and the position and size of fluorochrome bands and 45 rDNA FISH signals. A, B, C, D indicate M. sinensis, M. floridulus, M. sacchariflorus and M. lutarioriparius, respectively. The ordinate scale on the left indicates the relative length of the chromosomes (i.e.% of haploid complement). The numerical values under each chromosome pair indicate the arm ratios of the respective chromosome pair. The numbers above panel A are chromosome numbers.

All the four Miscanthus species had diploid chromosome number of 2n = 2x = 38. The mitotic metaphase chromosomes with a mean chromosome length 3.59 μm for M. sacchariflorus and 4.53 μm for M. floridulus. The total length of the haploid complement (TCL) ranges from 68.15 μm to 86.13 μm, and the mean centromeric index (CI) of the complements varied slightly between 44.00 ± 4.97 and 44.81 ± 4.28. In contrast, M. floridulus has exhibited the large variation in chromosome length, whereas M. sinensis has displayed the large variation in centromeric index.

The karyotype formulas of the four Miscanthus species were same, composed of 34 metacentric (m) chromosomes and 4 submetacentric (sm) chromosomes with a secondary constriction located on the long arms of chromosome 1, namely 2n = 2x = 38 = 34m(2SAT) + 4sm. All the karyotypes of the four species studied fell into the categories 2B of Stebbins (1971). The ranges for Romero Zarco’s (1986) asymmetry indices were as follows: A1 = 0.11–0.20 and A2 = 0.24–0.52. The As K% of Arano (1963) ranged from 55.25 to 55.85, and Paszko’s (2006) asymmetry index (AI) ranged from 2.37 to 4.47. RRL, CI, A1 and As K% have shown close similarity among species. In contrast, TCL, A2, and AI have displayed relatively large variation among species. According to the AI values, the karyotype of M. sacchariflorus is the most symmetrical and that of M. lutarioriparius is the most asymmetrical among the four species.

After CPD staining, slightly different fluorochrome banding patterns were observed among the four Miscanthus species (Fig. 1–3; Table 3). The red CPD bands were recorded in all species, whereas blue DAPI bands were found only in M. sinensis and M. floridulus.

Results showed that only one pair of CPD bands in M. sinensis, M. sacchariflorus and M. lutarioriparius had occurred in the secondary constrictions on the long arms of chromosome 1, and were co-localized with the 45S rDNA-FISH hybridization sites (called rDNA CPD bands; Fig. 1A, E, G). There were three CPD bands in the M. floridulus: two bands correspond to the secondary constriction on the long arms of chromosome 1; the other band was a non-rDNA CPD band with weaker fluorescence, occurring in the pericentromeric region of a homologue of chromosome pair 15 (Fig. 1G). The rDNA CPD bands in M. sinensis and M. lutarioriparius were similar in size and intensity on the two homologous chromosomes, while those in M. floridulus and M. sacchariflorus displayed heterozygosity, the band on one chromosome was large and bright, whereas the band on the other homologue was small and weak. The CPD bands of M. sinensis, M. floridulus, M. sacchariflorus and M. lutarioriparius accounted for 0.93%, 1.11%, 0.90% and 0.75% of the karyotype length, respectively.

M. sinensis showed seven blue DAPI bands (Fig. 1A, 2A, 3A): two pairs of weak bands occurred in the centromeric regions of chromosomes 10 and the pericentromeric regions of the long arm of chromosome 12, three relatively strong bands occured on chromosome 19. Among the DAPI bands on pair 19, two were located in the centromeric regions of both homologues, and one occured in the proximal region of the long arm of one homologue. M. floridulus had shown only one pair of weak DAPI bands in the pericentromeric regions on the chromosomes 19 (Fig. 1G, 2B, 3B). The DAPI bands of M. sinensis and M. floridulus accounted for 1.94% and 0.45% of the karyotype length, respectively.

45S rDNA FISH showed that M. sinensis, M. floridulus, M. sacchariflorus and M. lutarioriparius had only one pair of 45S rDNA sites, which were located in the secondary constriction on the long arms of chromosome 1, and their percentage distances of 45S rDNA sites were 25.53 ± 1.17, 29.43 ± 1.12, 32.07 ± 0.49, 28.45 ± 0.89, respectively. The 45S rDNA sites of the four Miscanthus species corresponded to their respective CPD bands in both size and intensity, that is, the 45S rDNA signals of two homologues in M. sinensis and M. lutarioriparius were similar in size and intensity, while those in M. floridulus and M. sacchariflorus differed in size and intensity, displaying heterozygosity.

The GISH results are shown in Fig. 4. Both self-GISH (sGISH; the genomic DNA of a species is applied to its own chromosomes) and cGISH generated hybridization signals in most regions of all chromosomes. Overall, the hybridization signals in the proximal and/or centromeric regions of the chromosomes were strong or very strong, while those in the proximal regions were relatively weak. In the GISHs to the M. sinensis chromosomes, the signals generated by M. floridulus gDNA (Fig. 4B) were stronger and more evenly distributed than those generated by M. sacchariflorus and M. lutarioriparius gDNAs (Fig. 4C, D), and more similar to the sGISH signals of M. sinensis (Fig. 4A). The hybridization signals of M. floridulus gDNA to the chromosomes of M. sacchariflorus (Fig. 4F) and M. lutarioriparius (Fig. 4I) were weaker than both the sGISH signals of M. sacchariflorus (Fig. 4E) and M. lutarioriparius (Fig. 4H), and the cGISH signals of M. lutarioriparius gDNA to M. sacchariflorus chromosomes (Fig. 4G) .

Figure 4.

FISH with M. sinensis (A), M. floridulus (B), M. sacchariflorus (C) and M. lutarioriparius (D) genomic DNA probes (red) to M. sinensis chromosomes, FISH with M. sacchariflorus (E), M. floridulus (F) and M. lutarioriparius (G) genomic DNA probes (red) to M. sacchariflorus chromosomes, and FISH with M. lutarioriparius (H) and M. floridulus (I) genomic DNA probes (red) to M. lutarioriparius chromosomes. Scale bars: 10 μm.

Each ITS1-5.8S-ITS2 sequences were compared to the published sequences of Miscanthus and its related species, and the boundaries of the spacer regions were confirmed. The length and other characteristics of each ITS1-5.8S-ITS2 are given in Table 4. The entire ITS sequence (ITS1-5.8S-ITS2) of I. cylindrica that was used as the outgroup species was 684 bp in length, and its GC content was 63.89%.

Neighbour joining (NJ) and maximum likelihood (ML) phylogenetic trees were developed based on the entire ITS sequences. The NJ and ML trees were very similar (Fig. 5), and the four Miscanthus species were divided into two categories: (i) group I contained M. sinensis and M. floridulus, which resulted in 82% (NJ) and 94% (ML) bootstrap values; (ii) group II included M. sacchariflorus and M. lutarioriparius, with 83% and 84% bootstrap values in NJ and ML trees, respectively. It was worth noting that in each branch the accessions of one species were not separated from those of another species (Fig. 5).

Figure 5.

Rooted neighbour-joining (NJ) (A) and maximum-likelihood (ML) (B) tree based on the ITS1-5.8S-ITS2 sequences of the four Miscanthus species using the MEGA software (version 7.0) with I. cylindrica as an outgroup species. The numbers near the nodes indicate bootstrap values (in percentage).

In the present study, detailed karyotypes of M. sinensis, M. floridulus, M. sacchariflorus and M. lutarioriparius were established using a combination of chromosome measurements, fluorochrome bands and 45S rDNA FISH signals, which provided the primary molecular-cytogenetic characterization of the four Miscanthus energy plants for the first time.

Our results had shown that the molecular cytogenetic karyotypes were rather similar among the four Miscanthus species. For instance, their karyotype formula, the categories of Stebbins, and the number and location of the 45S rDNA sites were same and there were slight differences in RRL, CI, A1 and As K%. However, several differences in their karyotypes were recorded: (1) obvious differences in their TCL, A2 and AI. In particular, AI, which can more accurately reveal the heterogeneity of chromosome length and centromere index in karyotype (Paszko 2006), had significant differences among the four species. (2) A non-rDNA CPD band appeared in M. floridulus. (3) AT-rich heterochromatin (DAPI bands) occurred in M. sinensis and M. floridulus but not in M. sacchariflorus and M. lutarioriparius. (4) There appeared 45S rDNA heterozygosity in M. floridulus and M. sacchariflorus but not in M. sinensis and M. lutarioriparius. Therefore, the four Miscanthus species could be accurately distinguished from each other using the molecular-cytogenetic karyotypic data.

As our study revealed, the chromosome numbers of the four Miscanthus species were all 2n = 2× = 38, being consistent with those reported previously (Hodkinson et al. 2002a, Takahashi and Shibata 2002, Takahashi et al. 2002, Chramiec-Głąbik et al. 2012). The current karyotypes of the four Miscanthus species comprised mainly of metacentric chromosomes, differing from the previous karyotypes, which had more submetacentric chromosomes, and even had acroentric chromosomes (Chramiec-Głąbik et al. 2012). Our study revealed that each species had a pair of satellite chromosomes, which were designated as chromosome 1. Previous studies also reported the presence of satellite chromosomes in Miscanthus species and revealed by FISH one pair of 45S rDNA sites in M. sinensis, but the serial number of the satellite chromosomes and the locations of the secondary constrictions were different (Takahashi and Shibata 2002, Takahashi et al. 2002, Chramiec-Głąbik et al. 2012). The deviations in karyotype were probably mainly due to differences in the material analysed, and difficulty in accurately pairing homologous chromosomes and distinguishing chromosomes by using the classical staining technique as applied before.

The research on the evolutionary relationship among the four Miscanthus species could not be conducted using genomic information because the genome sequences of M. floridulus, M. sacchariflorus and M. lutarioriparius were currently unavailable. Therefore, at present the combination of molecular cytogenetic analysis with rDNA ITS and chloroplast DNA sequence analysis was an effective phylogenetic analysis pathway (She et al. 2015).

As mentioned above, the molecular cytogenetic karyotypes of the four Miscanthus species were very similar, indicating the high genome similarity and small genome differentiation among them. Furthemore, the cGISH signals were rather similar in intensity and distribution to the sGISH signals, further demonstrating the high similarity among the four Miscanthus genomes (Wolny and Hasterok 2009, She et al. 2015, Zhang et al. 2015). However, the differences in molecular cytogenetic karyotypes and cGISH signals among the four species also provided valuable evolution information: (1) AT-rich heterochromatin appeared in both M. sinensis and M. floridulus, but did not emerge in both M. sacchariflorus and M. lutarioriparius; (2) The hybridization signals of M. floridulus gDNA to M. sinensis chromosomes were stronger and more evenly distributed than those of M. sacchariflorus and M. lutarioriparius gDNA, whereas the hybridization signals of M. floridulus gDNA to M. sacchariflorus chromosomes were weaker than the hybridization signals of M. lutarioriparius gDNA to M. sacchariflorus chromosomes. This information indicates that there was a close phylogenetic relationship between M. sinensis and M. floridulus, and between M. sacchariflorus and M. lutarioriparius; the former two species were relatively distant from the latter two species.

In our study, the phylogenetic tree based on the ITS sequences had shown that, M. sinensis and M. floridulus clustered into one branch, and M. sacchariflorus and M. lutarioriparius clustered into another branch. It was consistent with not only the above molecular cytogenetic results, but also the previous clustering results based on morphology, molecular markers and the ITS sequence (Hodkinson et al. 2002b, Chen et al. 2007, Sun et al. 2010, Chae et al. 2014, Clark et al. 2014, Ge et al. 2017). In addition, our clusering analysis revealed that the accessions of the two species in each branch were mixed and without distinct boundaries. These findings were consistent with the phylogenetic trees of Miscanthus and related genera inferred from ITS sequences (Hodkinson et al. 2002b, Chen et al. 2007, Liu et al. 2010), SSR markers (Ge et al. 2017), SNPs (Clark et al. 2014) and the dataset of genome size, ploidy level and genomic polymorphisms (Chae et al. 2014). To summarise, our clustering results have demonstrated that there was a very close phylogenetic relationship between M. sinensis and M. floridulus, and between M. sacchariflorus and M. lutarioriparius and they cannot be distinguished only based on the ITS sequences. However, as revealed in this study, the molecular cytogenetic karyotype analysis can effectively identify the four species.

Molecular cytogenetic karyotypes of M. sinensis, M. floridulus, M. sacchariflorus and M. lutarioriparius were established for the first time, which can effectively distinguish the four species. Molecular cytogenetic comparison revealed basic similarities and certain differences in genome organization among the four species. These findings will provide a reference for further development of specific probes based on M. sinensis genome sequence for chromosomal localization in the species of Miscanthus and related genera. The combined data of molecular cytogenetic and ITS sequence analysis indicated a close phylogenetic relationship between M. sinensis and M. floridulus, and between M. sacchariflorus and M. lutarioriparius, respectively. It can be concluded that former two species have relatively distant relationship compared with the latter two species.