Next Article in Journal
Meta-Analysis of Factors Affecting C-N Fractions and Yield of Paddy Soils by Total Straw Return and N Fertilizer Application
Previous Article in Journal
Dissipation and Dietary Risk Assessment of Thiacloprid and Tolfenpyrad in Tea in China
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 12
Issue 12
10.3390/agronomy12123167
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Identification of Sugarcane S. spontaneum (Poaceae) Germplasm: Evidence from rDNA-ITS and rDNA Locus Analyses

by
Pingping Lin
1,†,
Xuguang Hu
2,†,
Li Xue
1,
Xinyi Li
1,
Ping Wang
2,
Xinwang Zhao
2,
Muqing Zhang
1,
Zuhu Deng
1,2,* and
Fan Yu
1,*
1
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi Key Laboratory for Sugarcane Biology, Guangxi University, Nanning 530004, China
2
Key Lab of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2022, 12(12), 3167; https://doi.org/10.3390/agronomy12123167
Submission received: 18 November 2022 / Revised: 30 November 2022 / Accepted: 13 December 2022 / Published: 14 December 2022
(This article belongs to the Section Crop Breeding and Genetics)
Browse Figures
Review Reports Versions Notes

Abstract

:
Sugarcane is a major crop for sugar production around the world. The complexity of the sugarcane genome creates challenges for the use of both conventional and molecular breeding methods to improve sugarcane at a genetic level. DNA sequencing is an important tool to assess how the genus Saccharum and the genera of the Saccharum complex are interrelated. Here, we identify the kinship of Nepal2013-6 (Saccharum spontaneum, x = 10) using a tetra-primer amplification refractory mutation system (ARMS) PCR. Based on rDNA-ITS sequence analysis, the accession Nepal2013-6 falls within a single cluster with S. spontaneum (Yunnan82-114 and SES208), which is consistent with the previous results. Moreover, fluorescence in situ hybridization (FISH) results indicate that the 5S rDNA spots are consistent with the chromosomal ploidy in the analytical Saccharum materials, whereas 35S rDNA has similar or fewer sites than the ploidy. Therefore, 5S rDNA FISH patterns would be more suitable than 35S rDNA for chromosomal ploidy analysis in S. spontaneum with varied basic chromosome number x = 8, 9, 10. Altogether, these results indicate that the rDNA sequences will be a useful marker for further rapidly identifying the relationship and ploidy of S. spontaneum in sugarcane breeding.

1. Introduction

Sugarcane (Poaceae) belongs to the genus Saccharum L. and provides around 80% of the sugar produced worldwide. Sugarcane is also a feedstock crop for the production of biofuels, particularly ethanol, due to its high lignocellulosic biomass content [1,2]. Thus, sugarcane has significant economic value and is extensively cultivated. Saccharum taxonomically comprises six species, including S. spontaneum, S. robustum, S. officinarum, S. sinense, S. barberi, and S. edule. Among them, S. sinense, S. barberi, and S. edule were likely formed through interspecific or intergeneric hybridization [3]. Hence, two wild species, S. spontaneum and S. robustum, together with S. officinarum, are the founding species of Saccharum [4]. Modern sugarcane cultivars are hybrids in which S. officinarum, through nobilization, is introgressed with the desirable agronomic traits of S. spontaneum, such as stress tolerance and biomass accumulation [5,6]. Hence, S. spontaneum is one of the most valuable germplasm resources for innovation in sugarcane breeding.
S. spontaneum is a complex Saccharum species that is characterized by variable chromosome numbers (2n = 40 to 2n = 128) [7]. D’Hont et al. preliminarily determined that for S. spontaneum, the basic chromosome number is x = 8, whereas both S. officinarum and S. robustum have x = 10 based on the location of the ribosomal DNA (rDNA) [8]. Moreover, a series including 2n = 40, 48, 56, 64, 72, 80, 88, 96, 112, 120, and 128 accounts for 77% of the data reported for S. spontaneum, suggesting that its germplasm is a polyploid Saccharum with a basic number of x = 8. Interestingly, a recent study in which S. spontaneum chromosomes were aligned with those of sorghum revealed that chromosome fission and fusion reduced the S. spontaneum basic chromosome number from 10 to 8 [5,9,10]. Furthermore, several studies showed that S. spontaneum had a varied basic chromosome number, x = 9 and 10 [11,12,13]. Hence, a highly effective chromosome marker is urgently needed to identify the relationship and ploidy of the S. spontaneum germplasm for sugarcane genetic improvement.
rDNA internal transcribed spacers (ITS) are thought to evolve at a faster rate compared with other coding regions [14]. Thus, ITS sequences could be more valuable for the investigation and determination of Saccharum and its related genera [15,16]. Indeed, Liu et al. revealed phylogenetic relationships between Saccharum and its related genera through the analysis of ITS sequences [17]. Yang et al. analyzed ITS sequences from the founding species of Saccharum to identify a stable base mutation that was present in S. spontaneum with x = 8, but not detected in S. spontaneum with x = 10. They then developed tetra-primer ARMS PCR technology, which can effectively identify S. spontaneum (x = 8) germplasms based on this stable single nucleotide polymorphism (SNP) [18]. These studies demonstrate that the ITS region is a reliable molecular marker not only for phylogenetic analyses but also for the identification of germplasms in the genus Saccharum. Based on previous studies on rDNA-ITS in sugarcane germplasms, the 5S and 35S rDNA can be used to analyze chromosome evolution, in the detection of chromosome ploidy, and for karyotype analysis in plants. However, there is still lack of effective markers for the rapid identification of the recently discovered novel S. spontaneum germplasm with x = 10. In this study, we cloned, sequenced, and tested the tetra-primer amplification refractory mutation system (ARMS) PCR in Saccharum or its related genera to explore whether the rDNA will be applicable. These results provide a foundation for improving the efficiency of the relationship and chromosome ploidy identification of S. spontaneum for sugarcane genetic resource innovation.

2. Materials and Methods

2.1. Plant Material and DNA Extraction

Six taxa of the Saccharum complex were used in this study (Table 1). Genomic DNA was extracted from young leaves using a previously described cetyltrimethyl ammonium bromide (CTAB) procedure with minor modifications [19].

2.2. Molecular Cloning and Sequencing

The rDNA-ITS sequences (including ITS1, 5.8S rDNA, and ITS2) were amplified by polymerase chain reaction (PCR) with the universal primer pair ITS1 and ITS4 (ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′; ITS4: 5′-TCCTCCGCTTATTGATATGC-3′) [20]. Each PCR reaction mixture contained 50 ng genomic DNA, 2 μL 10× LA Buffer II (with Mg2+ plus), 1.6 μL dNTP mixture (2.5 mM each), 8 μM of each primer, and 0.2 μL LA Taq polymerase (Takara, Bio Inc., Tokyo, Japan) in 20 μL. PCR was carried out in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Waltham, MA, USA), with 1 cycle of pre-denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 20 s, annealing at 54 °C for 15 s, extension at 72 °C for 10 s, and a final extension at 72 °C for 5 min. Subsequently, the reaction products were isolated from a 1.5% agarose gel and purified using a QIAquick Gel Extraction Kit (Takara, Bio Inc., Tokyo, Japan). The purified fragments were ligated into the pMD19-T-vector (Takara, Bio Inc., Tokyo, Japan) and the constructs were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China).

2.3. Sequence Analysis and Tetra-Primer ARMS PCR Procedure

Data matrices of ITS sequences that included three taxa of the Solanum genus as an outgroup, and six taxa from the Saccharum complex, including genera Saccharum, Tripidium, Miscanthus, and Narenga, were used for phylogenetic analyses. Accordingly, the sequences were aligned using the “Align by ClustalW” feature in the MEGA 6.0 software [21]. The phylogenetic tree was constructed using the maximum likelihood (ML) method, and all clades were assessed with 1000 bootstrap replications. The ITS sequences of genus Saccharum and Nepal2013-6 were simultaneously analyzed using DNAMAN 7.0 to assess sequence similarity and variable sites. Tetra-primer ARMS PCR was performed according to the method established by Yang et al. [18] and reaction products were detected by 1.5% agarose gel electrophoresis.

2.4. Determination of Basic Chromosome Numbers by FISH Location with 5S and 35S rDNA

Slides were prepared as described in Huang et al. [22]. The 35S rDNA was based on probe pTA71 plasmids from wheat [23], labeled by nick translation with digoxigenin (Dig)-11-dUTP (Roche Co., Ltd., Indianapolis, IN, USA). The 5S rDNA probe was labeled with biotin-dUTP by PCR amplification using a pair of specific primers (5S-U: 5′-TCCTGGGAAGTCCTCGTGTTGCAT-3′ and 5S-L: 5′-GGTCACCCATCCTAGTACTACTCT-3′). For hybridization, chromosomes were denatured in 70% formamide with 2× SSC at 75 °C 2 min. The slides were then immediately dehydrated in a series of cold ethanol washes (70, 95, and 100%) for 5 min each. A 30 μL hybridization mixture containing 3 μL of each probe, 15 μL deionized formamide, 6 μL dextran sulfate, and 3 μL 2× SSC was incubated overnight at 37 °C. Stringency washes were performed as follows: once in 2× SSC at 42 °C for 10 min, twice in 4× SSC with 0.2% Tween-20 at 42 °C for 5 min, and once in 1× PBS at room temperature for 5 min. Digoxigenin and biotin-labeled probes were detected by rhodamine anti-Dig-sheep (Roche Co., Ltd., Indianapolis, IN, USA) and Alexafluor 488 Streptavidin (Life Technologies, Carlsbad, CA, USA), respectively. Finally, slides were counterstained with 4′,6′-diamidino-phenylindole (DAPI) in a Vectashield anti-fade solution (Vector Laboratories, Burlingame, CA, USA). FISH signals were examined under an AxioScope A1 Imager fluorescent microscope (Carl Zeiss, Gottingen, Germany). Images were captured with an AxioCam MRc5 and analyzed using the AxioVision v. 4.7 imaging software.

3. Results

3.1. Detection of SNP Sites and Tetra-Primer ARMS PCR

Alignment of the ITS sequences indicated the presence of a stable base mutation among S. spontaneum, S. officinarum, and S. robustum (Figure 1). These SNPs were identical to those seen by Yang et al. [18]. Tetra-primer ARMS PCR was then performed to identify Nepal2013-6. Two bands, 428 bp and 203 bp, were seen for Yunnan82-114 (x = 8), SES208 (x = 8), and Nepal2013-6 (x = 10) (Figure 2). Meanwhile, S. officinarum (Badila-CN, LA Purple) and S. robustum (51NG63 and 57NG208) had a common 428 bp band and a specific band at 278 bp (Figure 2). These results indicated that the base mutation of the ITS sequences of S. spontaneum x = 10 (Nepal2013-6) was conserved and could be used for the identification of the relationship of S. spontaneum with the basic chromosome x = 10 in sugarcane breeding.

3.2. Analysis of rDNA-ITS

The rDNA-ITS region was PCR-amplified and sequenced for at least five individual plants of each species, and a single 678 bp product was obtained. The ITS sequences were aligned and the maximum likelihood method was used to construct a phylogenetic tree. Phylogenies were well resolved, where, in the Saccharum complex species, there was a single clade and it had high Bayesian support values in nodes (Figure 3). As predicted, Saccharum, Tripidium, Miscanthus, and Narenga formed four obvious groups (Figure 3), indicative of four different genera in the Saccharum complex. Interestingly, Nepal2013-6, Yunnan82-114 (S. spontaneum), and SES208 (S. spontaneum) fell within a single cluster, while S. officinarum (Badila-CN and LA Purple) and S. robustum (51NG63 and 57NG208) fell within another cluster. These results indicate that rDNA-ITS sequences also could be used for the phylogenetic analysis of S. spontaneum with the basic chromosome x = 10.

3.3. Physical Mapping of 5S and 35S rDNAs in the Genus Saccharum

Identifying the chromosomal locations of rDNA can increase our understanding of ploidy, which would be important for research concerning plant evolution—particularly in S. spontaneum, which had many chromosome numbers, 2n = 40–128, and varied basic chromosomes x = 8, 9, 10. To investigate the chromosomal distribution of the 5S and 35S rDNA in the genus Saccharum, FISH analysis was performed using biotin-labeled 5S rDNA and Dig-labeled 35S rDNA as probes. In these experimental materials, the chromosomal ploidy was consistent with the number of 5S rDNA loci, but was equal to or less than that for 35S rDNA loci in S. officinarum and S. robustum (Figure 4, Table 2).
Additionally, both the 5S and 35S rDNA probes produced four obvious signals that were located in the pericentromeric and distal chromosomal regions, respectively, in Nepal2013-6 (Figure 5). This result supports a basic chromosome number for Nepal2013-6 of x = 10, which differs from the classic x = 8 in S. spontaneum. Furthermore, FISH results indicated that all 35S rDNA loci (S. spontaneum, x = 8) were located in the subtelomere region in S. spontaneum (x = 8) (Figure 6), which differs from S. spontaneum (x = 10), S. officinarum, and S. robustum (telomere, Figure 4). Taken together, these results indicated that the 35S rDNA loci were more varied than those for 5S rDNA for the purpose of determining the basic chromosome number and ploidy in the genus Saccharum, especially in S. spontaneum (Table 2).

4. Discussion

Due to the higher variability relative to flanking coding regions [24], ITS sequences have been widely applied for the study of phylogenetic relationships in plants and animals [25,26,27,28]. Moreover, the rDNA-ITS sequence evolves more rapidly than the flanking coding regions and thus these sequences have been widely applied to assess intra-species variation, which has allowed the identification of closely related species within families and even within genera [29]. Data for rDNA-ITS phylogenetic analysis supported the presence of different genera among Saccharum, Tripidium, Miscanthus, and Narenga within the Saccharum complex [30]. Notably, Nepal2013-6 fell within S. spontaneum, in the same clade that includes Yunnan82-114 and SES208.
The Saccharum complex includes five genera and is characterized by different, high polyploidy levels [31,32]. Distinguishing Saccharum germplasms based on morphology is very challenging. Thus, it is necessary to find and develop new molecular markers that are stable in heredity and free from environmental deviation. Molecular marker methods such as AFLP, SSR, ISSR, and RAPD have recently been used as conventional techniques for the identification of germplasms and their progeny [33,34,35,36,37,38,39]. However, all of these methods are based on the presence of multiple amplification bands for the identification of species, which can produce variable results for the authentication of germplasms in sugarcane. Tetra-primer ARMS PCR showed that there is a stable mutation at base 89 in the rDNA-ITS sequence of the genus Saccharum. This mutation has since been successfully applied to identify S. spontaneum (x = 8) genetic signatures in the genus Saccharum [18]. In this study, tetra-primer ARMS PCR was used to identify S. spontaneum (x = 10) based on a stable SNP of rDNA-ITS and was consistent with the phylogenetic analysis of the ITS sequences. These results revealed that rDNA-ITS sequences also can be used in identifying the relationship of S. spontaneum with x = 10, which will promote the utilization of S. spontaneum x = 10 in sugarcane breeding.
The 5S and 35S rDNA are widely used markers for FISH studies on chromosome evolution, for the detection of chromosome ploidy, and for karyotype analysis in plants [8,40]. For example, ribosomal DNA probes were used to confirm chromosome ploidy in S. officinarum and S. spontaneum [8]. In theory, the number of rDNA loci should be equal to the autopolyploid ploidy in most Poaceae plants. Thus, the 35S and 5S rDNA loci numbers are often adopted to determine the basic chromosome numbers of species through FISH location. However, some studies showed that the number of 35S rDNA loci can differ from chromosomal ploidy due to the dynamic evolution of 35S rDNA [8,41]. Considering this potential instability of 35S rDNA loci, 5S rDNA would be more suitable for the determination of basic chromosome numbers. Moreover, most 35S rDNA has a terminal location on the chromosome, whereas 5S rDNA is generally located at pericentromeric positions within species [42,43]. Thus, the location of 5S rDNA is likely more conserved than that of 35S rDNA and may result in polymorphism of the 35S rDNA sites in many species. As shown here, the number of 5S rDNA loci in the genus Saccharum was consistent with the chromosomal ploidy. Meanwhile, 35S rDNA loci showed polymorphisms among different ploidy species. Hence, 5S rDNA FISH patterns would be more suitable than 35S rDNA for chromosomal ploidy analysis in the genus Saccharum. Of these, S. spontaneum provides a good biological genetic background, such as tolerance and strong adaptability. As a valuable germplasm resource, S. spontaneum has made a large contribution to sugarcane breeding. However, compared to S. officinarum and S. robustum, with x = 10, S. spontaneum has a varied basic chromosome number, x = 8, 9, and 10 [11,12,13]. Hence, it will be very important to develop a marker with a low cost and time-saving benefits for the identification of the ploidy of S. spontaneum. Our results for the loci locations of both 5S and 35S rDNA indicated that 5S rDNA will be efficient for the identification of different ploidy in S. spontaneum.

5. Conclusions

In the present study, our results demonstrate that the rDNA-ITS region is a reliable molecular marker not only for phylogenetic analyses but also for the identification of germplasms in the genus Saccharum. Tetra-primer amplification refractory mutation system (ARMS) PCR amplification was proven to be useful for the identification of S. spontaneum with x = 10. A phylogenetic tree was constructed based on the rDNA-ITS sequences, which supported the presence of different genera among Saccharum, Tripidium, Miscanthus, and Narenga. Additionally, we performed FISH assays of the genus Saccharum with 5S and 35S rDNA probes to reveal the chromosomal pattern of rDNA in S. spontaneum with varied chromosomes. Altogether, these findings indicated that rDNA sequences will be efficient markers for the further rapid identification of the relationships and ploidy of S. spontaneum to obtain innovative genetic resources for sugarcane breeding.

Author Contributions

P.L., X.H. and F.Y. designed the research. P.L., X.H., L.X., X.L. and P.W. performed the experiments. P.L., X.H., L.X., X.L., X.Z., M.Z., Z.D. and F.Y. analyzed the results. P.L., X.H. and F.Y. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This project was supported by the Sugarcane Research Foundation of Guangxi University (No. 2022GZB006), an independent fund of the Guangxi Key Laboratory of Sugarcane Biology, and supported by the China Agriculture Research System of MOF and MARA (No. CARS-20-1-5). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the article.

Acknowledgments

We thank the Sugarcane Research Institute of Yunnan Agriculture Science Academy and the Research Institute Ruili Station of Yunnan Agriculture Science Academy for providing the plant materials used in this study. We also thank Bioscience Editing Solutions for critically reading this paper and providing helpful suggestions.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Goldemberg, J.; Coelho, S.T.; Guardabassi, P. The sustainability of ethanol production from sugarcane. Energy Policy 2008, 36, 2086–2097. [Google Scholar] [CrossRef]
  2. Wang, J.; Roe, B.; Macmil, S.; Yu, Q.; Murray, J.E.; Tang, H.; Chen, C.; Najar, F.; Wiley, G.; Bowers, J.; et al. Microcollinearity between autopolyploid sugarcane and diploid sorghum genomes. BMC Genom. 2010, 11, 261. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. D’Hont, A.; Paulet, F.; Glaszmann, J.C. Oligoclonal interspecific origin of ‘North Indian’ and ‘Chinese’ sugarcanes. Chromosome Res. 2002, 10, 253–262. [Google Scholar] [CrossRef]
  4. Zhang, J.; Zhang, Q.; Li, L.; Tang, H.; Zhang, Q.; Chen, Y.; Arrow, J.; Zhang, X.; Wang, A.; Miao, C.; et al. Recent polyploidization events in three Saccharum founding species. Plant Biotechnol. J. 2019, 17, 264–274. [Google Scholar] [CrossRef] [Green Version]
  5. Zhang, J.; Zhang, X.; Tang, H.; Zhang, Q.; Hua, X.; Ma, X.; Zhu, F.; Jones, T.; Zhu, X.; Bowers, J.; et al. Allele-defined genome of the autopolyploid sugarcane Saccharum spontaneum L. Nat. Genet. 2018, 50, 1565–1573. [Google Scholar] [CrossRef] [Green Version]
  6. Pastina, M.M.; Malosetti, M.; Gazaffi, R.; Mollinari, M.; Margarido, G.R.; Oliveira, K.M.; Pinto, L.R.; Souza, A.P.; van Eeuwijk, F.A.; Garcia, A.A. A mixed model QTL analysis for sugarcane multiple-harvest-location trial data. TAG Theor. Appl. Genet. Theor. Angew. Genet. 2012, 124, 835–849. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  7. Panje, R.R.; Babu, C.N. Studies in Saccharum spontaneum Distribution and Geographical Association of Chromosome Numbers. Cytologia 1960, 25, 152–172. [Google Scholar] [CrossRef] [Green Version]
  8. D’Hont, A.; Ison, D.; Alix, K.; Roux, C.; Glaszmann, J.C. Determination of basic chromosome numbers in the genus Saccharum by physical mapping of ribosomal RNA genes. Genome 1998, 41, 221–225. [Google Scholar] [CrossRef]
  9. Dong, G.; Shen, J.; Zhang, Q.; Wang, J.; Yu, Q.; Ming, R.; Wang, K.; Zhang, J. Development and Applications of Chromosome-Specific Cytogenetic BAC-FISH Probes in S. spontaneum. Front. Plant Sci. 2018, 9, 218. [Google Scholar] [CrossRef] [Green Version]
  10. Meng, Z.; Zhang, Z.; Yan, T.; Lin, Q.; Wang, Y.; Huang, W.; Huang, Y.; Li, Z.; Yu, Q.; Wang, J.; et al. Comprehensively Characterizing the Cytological Features of Saccharum spontaneum by the Development of a Complete Set of Chromosome-Specific Oligo Probes. Front. Plant Sci. 2018, 9, 1624. [Google Scholar] [CrossRef] [PubMed]
  11. Yu, F.; Zhao, X.; Chai, J.; Ding, X.; Li, X.; Huang, Y.; Wang, X.; Wu, J.; Zhang, M.; Yang, Q.; et al. Chromosome-specific painting unveils chromosomal fusions and distinct allopolyploid species in the Saccharum complex. New Phytol. 2022, 233, 1953–1965. [Google Scholar] [CrossRef] [PubMed]
  12. Zhang, Q.; Qi, Y.; Pan, H.; Tang, H.; Wang, G.; Hua, X.; Wang, Y.; Lin, L.; Li, Z.; Li, Y.; et al. Genomic insights into the recent chromosome reduction of autopolyploid sugarcane Saccharum spontaneum. Nat. Genet. 2022, 54, 885–896. [Google Scholar] [CrossRef] [PubMed]
  13. Piperidis, N.; D’Hont, A. Sugarcane genome architecture decrypted with chromosome-specific oligo probes. Plant J. Cell Mol. Biol. 2020, 103, 2039–2051. [Google Scholar] [CrossRef] [PubMed]
  14. Wang, C.T.; Wang, X.Z.; Tang, Y.Y.; Chen, D.X.; Cui, F.G.; Zhang, J.C.; Yu, S.L. Phylogeny of Arachis based on internal transcribed spacer sequences. Genet. Resour. Crop Evol. 2011, 58, 311–319. [Google Scholar] [CrossRef]
  15. Hsiao, C.; Chatterton, N.J.; Asay, K.H.; Jensen, K.B. Phylogenetic relationships of the monogenomic species of the wheat tribe, Triticeae (Poaceae), inferred from nuclear rDNA (internal transcribed spacer) sequences. Genome 1995, 38, 211–223. [Google Scholar] [CrossRef]
  16. De Bustos, A.; Jouve, N. Phylogenetic relationships of the genus Secale based on the characterisation of rDNA ITS sequences. Plant Syst. Evol. 2002, 235, 147–154. [Google Scholar] [CrossRef]
  17. Liu, X.-L.; Su, H.-S.; Ma, L.; Lu, X.; Ying, X.-M.; Cai, Q.; Fan, Y.-H. Phylogenetic Relationships of Sugarcane Related Genera and Species Based on ITS Sequences of Nuclear Ribosomal DNA. Acta Agron. Sin. 2010, 36, 1853–1863. [Google Scholar] [CrossRef]
  18. Yang, S.; Li, X.; Huang, F.; Huang, Y.; Liu, X.; Wu, J.; Wang, Q.; Deng, Z.; Chen, R.; Zhang, M. A new method based on SNP of nrDNA-ITS to identify Saccharum spontaneum and its progeny in the genus Saccharum. PLoS ONE 2018, 13, e0197458. [Google Scholar] [CrossRef] [Green Version]
  19. Stewart, C.N., Jr.; Via, L.E. A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. BioTechniques 1993, 14, 748–750. [Google Scholar]
  20. Besse, P. Nuclear Ribosomal RNA Genes: ITS Region. Methods Mol. Biol. 2021, 2222, 119–129. [Google Scholar] [CrossRef]
  21. Kumar, S.; Nei, M.; Dudley, J.; Tamura, K. MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief. Bioinform. 2008, 9, 299–306. [Google Scholar] [CrossRef] [PubMed]
  22. Huang, Y.; Luo, L.; Hu, X.; Yu, F.; Yang, Y.; Deng, Z.; Wu, J.; Chen, R.; Zhang, M. Characterization, Genomic Organization, Abundance, and Chromosomal Distribution of Ty1-copia Retrotransposons in Erianthus arundinaceus. Front. Plant Sci. 2017, 8, 924. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Gerlach, W.L.; Bedbrook, J.R. Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res. 1979, 7, 1869–1885. [Google Scholar] [CrossRef] [PubMed]
  24. Hillis, D.M.; Dixon, M.T. Ribosomal DNA: Molecular evolution and phylogenetic inference. Q. Rev. Biol. 1991, 66, 411–453. [Google Scholar] [CrossRef] [PubMed]
  25. Schlötterer, C.; Hauser, M.T.; von Haeseler, A.; Tautz, D. Comparative evolutionary analysis of rDNA ITS regions in Drosophila. Mol. Biol. Evol. 1994, 11, 513–522. [Google Scholar] [CrossRef] [Green Version]
  26. Garzón, C.D.; Yánez, J.M.; Moorman, G.W. Pythium cryptoirregulare, a new species within the P. irregulare complex. Mycologia 2007, 99, 291–301. [Google Scholar] [CrossRef]
  27. Coleman, A.W.; Vacquier, V.D. Exploring the phylogenetic utility of ITS sequences for animals: A test case for abalone (Haliotis). J. Mol. Evol. 2002, 54, 246–257. [Google Scholar] [CrossRef]
  28. Gurushidze, M.; Mashayekhi, S.; Blattner, F.R.; Friesen, N.; Fritsch, R.M. Phylogenetic relationships of wild and cultivated species of Allium section Cepa inferred by nuclear rDNA ITS sequence analysis. Plant Syst. Evol. 2007, 269, 259–269. [Google Scholar] [CrossRef]
  29. Yao, H.; Song, J.; Liu, C.; Luo, K.; Han, J.; Li, Y.; Pang, X.; Xu, H.; Zhu, Y.; Xiao, P.; et al. Use of ITS2 region as the universal DNA barcode for plants and animals. PLoS ONE 2010, 5, e13102. [Google Scholar] [CrossRef] [Green Version]
  30. Hodkinson, T.R.; Chase, M.W.; Lledó, M.D.; Salamin, N.; Renvoize, S.A. Phylogenetics of Miscanthus, Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnLintron and trnL-F intergenic spacers. J. Plant Res. 2002, 115, 381–392. [Google Scholar] [CrossRef]
  31. Ha, S.; Moore, P.H.; Heinz, D.; Kato, S.; Ohmido, N.; Fukui, K. Quantitative chromosome map of the polyploid Saccharum spontaneum by multicolor fluorescence in situ hybridization and imaging methods. Plant Mol. Biol. 1999, 39, 1165–1173. [Google Scholar] [CrossRef] [PubMed]
  32. D’Hont, A. Unraveling the genome structure of polyploids using FISH and GISH; examples of sugarcane and banana. Cytogenet. Genome Res. 2005, 109, 27–33. [Google Scholar] [CrossRef] [PubMed]
  33. Edmé, S.J.; Glynn, N.G.; Comstock, J.C. Genetic segregation of microsatellite markers in Saccharum officinarum and S. spontaneum. Heredity 2006, 97, 366–375. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Mary, S.; Nair, N.V.; Chaturvedi, P.K.; Selvi, A. Analysis of Genetic Diversity Among Saccharum spontaneum L. From Four Geographical Regions of India, Using Molecular Markers. Genet. Resour. Crop Evol. 2006, 53, 1221–1231. [Google Scholar] [CrossRef]
  35. Devarumath, R.M.; Kalwade, S.B.; Kawar, P.G.; Sushir, K.V. Assessment of Genetic Diversity in Sugarcane Germplasm Using ISSR and SSR Markers. Sugar Tech. 2012, 14, 334–344. [Google Scholar] [CrossRef]
  36. Chandra, A.; Grisham, M.P.; Pan, Y.B. Allelic divergence and cultivar-specific SSR alleles revealed by capillary electrophoresis using fluorescence-labeled SSR markers in sugarcane. Genome 2014, 57, 363–372. [Google Scholar] [CrossRef]
  37. Upadhyay, A.; Kadam, U.S.; Chacko, P.; Karibasappa, G.J.I.J.H. Microsatellite and RAPD analysis of grape (Vitis spp.) accessions and identification of duplicates/misnomers in germplasm collection. Indian J. Hortic. 2010, 67, 8–15. [Google Scholar] [CrossRef] [Green Version]
  38. Upadhyay, A.; Kadam, U.S.; Chacko, P.M.; Aher, L.; Karibasappa, G.S. Microsatellite analysis to differentiate clones of Thompson seedless grapevine. Indian J. Hortic. 2010, 67, 260–263. [Google Scholar] [CrossRef]
  39. Hinge, V.R.; Shaikh, I.M.; Chavhan, R.L.; Deshmukh, A.S.; Shelake, R.M.; Ghuge, S.A.; Dethe, A.M.; Suprasanna, P.; Kadam, U.S. Assessment of genetic diversity and volatile content of commercially grown banana (Musa spp.) cultivars. Sci. Rep. 2022, 12, 1–16. [Google Scholar] [CrossRef]
  40. Zhang, L.; Yang, X.; Tian, L.; Chen, L.; Yu, W. Identification of peanut (Arachis hypogaea) chromosomes using a fluorescence in situ hybridization system reveals multiple hybridization events during tetraploid peanut formation. New Phytol. 2016, 211, 1424–1439. [Google Scholar] [CrossRef]
  41. Huang, Y.; Yu, F.; Li, X.; Luo, L.; Wu, J.; Yang, Y.; Deng, Z.; Chen, R.; Zhang, M. Comparative genetic analysis of the 45S rDNA intergenic spacers from three Saccharum species. PLoS ONE 2017, 12, e0183447. [Google Scholar] [CrossRef] [PubMed]
  42. Singh, M.; Kumar, R.; Nagpure, N.S.; Kushwaha, B.; Gond, I.; Lakra, W.S. Chromosomal localization of 18S and 5S rDNA using FISH in the genus Tor (Pisces, Cyprinidae). Genetica 2009, 137, 245–252. [Google Scholar] [CrossRef] [PubMed]
  43. Vaio, M.; Speranza, P.; Valls, J.F.; Guerra, M.; Mazzella, C. Localization of the 5S and 45S rDNA sites and cpDNA sequence analysis in species of the Quadrifaria group of Paspalum (Poaceae, Paniceae). Ann. Bot. 2005, 96, 191–200. [Google Scholar] [CrossRef] [PubMed]
Agronomy 12 03167 g001 550
Figure 1. Alignment of partial internal transcribed spacers (ITS) sequences. Boxes indicate single nucleotide polymorphism (SNP) location within the genus Saccharum.
Figure 1. Alignment of partial internal transcribed spacers (ITS) sequences. Boxes indicate single nucleotide polymorphism (SNP) location within the genus Saccharum.
Agronomy 12 03167 g001
Agronomy 12 03167 g002 550
Figure 2. Tetra-primer ARMS PCR products. M: 100 bp marker. Lanes 1-7: Badila-CN, LA Purple, 51NG63, 57NG208, Yunnan82-114, SES208, Nepal2013-6, respectively.
Figure 2. Tetra-primer ARMS PCR products. M: 100 bp marker. Lanes 1-7: Badila-CN, LA Purple, 51NG63, 57NG208, Yunnan82-114, SES208, Nepal2013-6, respectively.
Agronomy 12 03167 g002
Agronomy 12 03167 g003 550
Figure 3. Phylogenetic tree of rDNA-ITS sequence that include three taxa of the Solanum genus as an outgroup, and six taxa from the Saccharum complex, including genera Saccharum, Tripidium, Miscanthus, and Narenga were used for phylogenetic analyses. Bootstrap values over 50% after 1000 replications are indicated at the nodes.
Figure 3. Phylogenetic tree of rDNA-ITS sequence that include three taxa of the Solanum genus as an outgroup, and six taxa from the Saccharum complex, including genera Saccharum, Tripidium, Miscanthus, and Narenga were used for phylogenetic analyses. Bootstrap values over 50% after 1000 replications are indicated at the nodes.
Agronomy 12 03167 g003
Agronomy 12 03167 g004 550
Figure 4. Localization of 5S rDNA and 35S rDNA probes on root–tip metaphase chromosomes of S. officinarum and S. robustum (2n = 8x = 80) by FISH. (A–D): Badila-CN; LA Purple; 51NG63; 57NG208, respectively. The 5S rDNA signals are green and indicated by white arrows. The 35S rDNA signals are red and indicated by yellow arrows. Scale bars = 5 μm.
Figure 4. Localization of 5S rDNA and 35S rDNA probes on root–tip metaphase chromosomes of S. officinarum and S. robustum (2n = 8x = 80) by FISH. (A–D): Badila-CN; LA Purple; 51NG63; 57NG208, respectively. The 5S rDNA signals are green and indicated by white arrows. The 35S rDNA signals are red and indicated by yellow arrows. Scale bars = 5 μm.
Agronomy 12 03167 g004
Agronomy 12 03167 g005 550
Figure 5. Localization of 5S rDNA and 35S rDNA probes on root–tip metaphase chromosomes of Nepal2013-6 (2n = 4x = 40) by FISH. The 5S rDNA signals are green and indicated by white arrows. The 35S rDNA signals are red and indicated by yellow arrows.
Figure 5. Localization of 5S rDNA and 35S rDNA probes on root–tip metaphase chromosomes of Nepal2013-6 (2n = 4x = 40) by FISH. The 5S rDNA signals are green and indicated by white arrows. The 35S rDNA signals are red and indicated by yellow arrows.
Agronomy 12 03167 g005
Agronomy 12 03167 g006 550
Figure 6. Localization of 5S rDNA and 35S rDNA probes on root–tip metaphase chromosomes of S. spontaneum by FISH. (A–H): SES208; Yunnan82-63; Yunnan83-160; Sichuan92-42; Yunnan82-50; Yunnan82-114; Yunnan83-171; Guizhou78-2-28, respectively. The 5S rDNA signals are green and indicated by white arrows. The 35S rDNA signals are red and indicated by yellow arrows.
Figure 6. Localization of 5S rDNA and 35S rDNA probes on root–tip metaphase chromosomes of S. spontaneum by FISH. (A–H): SES208; Yunnan82-63; Yunnan83-160; Sichuan92-42; Yunnan82-50; Yunnan82-114; Yunnan83-171; Guizhou78-2-28, respectively. The 5S rDNA signals are green and indicated by white arrows. The 35S rDNA signals are red and indicated by yellow arrows.
Agronomy 12 03167 g006
Table
Table 1. Plant materials used in this study.
Table 1. Plant materials used in this study.
No.CloneSpeciesPloidyChromosome NumberAnalysis
1Badila-CNS. officinarum880rDNA-ITS and locus
2LA PurpleS. officinarum880rDNA-ITS and locus
351NG63S. robustum880rDNA-ITS and locus
457NG208S. robustum880rDNA-ITS and locus
5SES208S. spontaneum864rDNA-ITS and locus
6Yunnan82-63S. spontaneum864rDNA locus
7Yunnan83-160S. spontaneum864rDNA locus
8Sichuan92-42S. spontaneum972rDNA locus
9Yunnan82-50S. spontaneum972rDNA locus
10Yunnan82-114S. spontaneum1080rDNA-ITS and locus
11Yunnan83-171S. spontaneum1080rDNA locus
12Guizhou78-2-28S. spontaneum1296rDNA locus
13Nepal2013-6S. spontaneum440rDNA-ITS and locus
14Hainan92-77Tripidium arundinaceum660rDNA-ITS
15Hainan92-105Tripidium arundinaceum660rDNA-ITS
16Jiangxi91-8Miscanthus sinensis438rDNA-ITS
17Yunnan95-9Miscanthus sinensis438rDNA-ITS
18Guangdong64Narenga pophyrocoma430rDNA-ITS
19Sichuan92-11Narenga pophyrocoma430rDNA-ITS
Table
Table 2. The 5S and 35 rDNA loci in S. officinarum, S. robustum, and S. spontaneum.
Table 2. The 5S and 35 rDNA loci in S. officinarum, S. robustum, and S. spontaneum.
CloneOriginNo. of ChromosomesNo. of 5S rDNA LociNo. of 35S rDNA LociLocation Type
Badila-CN/2n = 8088telomere
LA Purple/2n = 8088telomere
51NG63/2n = 8086telomere
57NG208/2n = 8088telomere
Nepal2013-6Nepal2n = 4044telomere
SES208/2n = 6487subtelomere
Yunnan82-63Yunnan2n = 6486subtelomere
Yunnan83-160Yunnan2n = 6487subtelomere
Sichuan92-42Sichuan2n = 7298subtelomere
Yunnan82-50Yunnan2n = 7297subtelomere
Yunnan82-114Yunnan2n = 80106subtelomere
Yunnan83-171Yunnan2n = 80105subtelomere
Guizhou78-2-28Guizhou2n = 96128subtelomere
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Lin, P.; Hu, X.; Xue, L.; Li, X.; Wang, P.; Zhao, X.; Zhang, M.; Deng, Z.; Yu, F. Identification of Sugarcane S. spontaneum (Poaceae) Germplasm: Evidence from rDNA-ITS and rDNA Locus Analyses. Agronomy 2022, 12, 3167. https://doi.org/10.3390/agronomy12123167

AMA Style

Lin P, Hu X, Xue L, Li X, Wang P, Zhao X, Zhang M, Deng Z, Yu F. Identification of Sugarcane S. spontaneum (Poaceae) Germplasm: Evidence from rDNA-ITS and rDNA Locus Analyses. Agronomy. 2022; 12(12):3167. https://doi.org/10.3390/agronomy12123167

Chicago/Turabian Style

Lin, Pingping, Xuguang Hu, Li Xue, Xinyi Li, Ping Wang, Xinwang Zhao, Muqing Zhang, Zuhu Deng, and Fan Yu. 2022. "Identification of Sugarcane S. spontaneum (Poaceae) Germplasm: Evidence from rDNA-ITS and rDNA Locus Analyses" Agronomy 12, no. 12: 3167. https://doi.org/10.3390/agronomy12123167

APA Style

Lin, P., Hu, X., Xue, L., Li, X., Wang, P., Zhao, X., Zhang, M., Deng, Z., & Yu, F. (2022). Identification of Sugarcane S. spontaneum (Poaceae) Germplasm: Evidence from rDNA-ITS and rDNA Locus Analyses. Agronomy, 12(12), 3167. https://doi.org/10.3390/agronomy12123167

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop