Regular Article
Evolutionary Dynamics of Chromosomal Diversity in Saccharum spontaneum Population from Punjab and Haryana, India
2020 Volume 85 Issue 4 Pages 313-318
Details
2020 Volume 85 Issue 4 Pages 313-318
Saccharum spontaneum, the wild species of sugarcane, is an important germplasm resource with exceptional properties of pest and disease resistance and stress tolerance. Chromosomal diversity and its evolutionary dynamics in this species have been revealed through the study of 88 clones collected from the states of Punjab and Haryana, India. Twelve cytotypes were identified with five euploids [2n=40 (5x), 48 (6x), 56 (7x), 64 (8x), 72 (9x)] and seven aneuploids (2n=50, 52, 54, 60, 70, 74, 76). The random meiotic analysis showed predominant bivalent formation. The clone IND 16-1847 (2n=72) showed meiotic abnormalities like univalents, laggards, bridges, and multipolar chromosome segregation. Analysis of the evolutionary origin of different cytotypes revealed the independent, as well as multiple origins and the ploidy evolution, might be a network type rather than in a single direction. This leads to the increased allelic and genetic variation and gene flow among different ploidy levels of S. spontaneum.
Saccharum spontaneum L., is a wild species of sugarcane classified as the Andropogonea tribe of the Poacea family. This species contributed significantly to the varietal improvement in sugarcane and most of the modern sugarcane cultivars are complex interspecific hybrids involving S. spontaneum as one of the parents in their ancestry. S. spontaneum has strong environmental adaptability and contributes towards disease and other stress resistance in the modern sugarcane cultivars. This species is widely distributed in tropical and temperate regions worldwide and exhibits abundant morphological variation from short and bushy statured with short internodes to tall and erect with long internodes. S. spontaneum has originated in the Sub Himalayan regions of India and has the widest distribution in the country (Sreenivasan et al. 1987). It shows the extensive variation in cytotypes as well as in the morphological characters. The chromosome number in the species varies widely from 2n=40–128 and over all-around 30 cytotypes have been reported in the species (Panje and Babu 1960, Kandaswamy and Rao 1963, Sreenivasan et al. 2001, Sobhakumari and Mallika 2007, Sobhakumari 2009, Sobhakumari and Stanly, 2017). While analyzing the pedigree records of presently available commercial cultivars of sugarcane it is very clear that S. spontaneum contributed significantly to the varietal improvement in sugarcane and all the modern commercial cultivars are complex interspecific hybrids involving S. spontaneum as one of the parents in their ancestry. Apart from the pest and disease resistance and stress tolerance, the hardiness, adaptability, and high productivity of the sugarcane cultivars have been attributed to S. spontaneum complement of the hybrid genome.
A breakthrough in sugarcane improvement was achieved in sugarcane breeding programs by utilizing this wild species in hybridization. The first sugarcane cultivar, Co 205, developed at ICAR-Sugarcane Breeding Institute, Coimbatore, India, was an interspecific hybrid involving S. officinarum clone Vellai and S. spontaneum clone Coimbatore. This initial success in transferring desirable traits from S. spontaneum to cultivated sugarcane led to the systematic collection and conservation of these species from their original distributional areas. As a part of this activity, during 2016, 93 clones of S. spontaneum were collected from two western states of India, Punjab, and Haryana. After quarantine, these collections are clonally being maintained at the world germplasm collection of ICAR-Sugarcane Breeding Institute, Coimbatore and systematic characterization has been carried out.
This article reports the cytological characterization of 88 clones of S. spontaneum collected from the plains of two states, Punjab and Haryana, of the Northwestern part of India. The information on the chromosome number of different clones of S. spontaneum from this region was utilized to work out the process underlying chromosomal evolution within the species. This information will be useful for the effective utilization of these clones in the sugarcane improvement program.
The cytological survey included 88 clones of S. spontaneum (IND 16 accessions) collected from different altitudes of North-western states, Punjab and Haryana, of India. These clones were collected during 2016. Small clumps of these clones were taken from the germplasm field and planted in pots and root tips were collected between 1 : 00–1 : 30 p.m. and pre-treated with a saturated solution of α-bromo naphthalene for 2 h at 4°C. The roots were then washed in running water and fixed in ethyl alcohol : acetic acid (3 : 1) solution and kept in a refrigerator overnight. The root tips were washed and hydrolyzed in 1 N HCl for 13 min at 60°C and stained in leuco basic fuchsin for 30 min and squashed in 1% acetocarmine. A minimum of 10 well-spread metaphase plates was used for chromosome count and photographed in a Carton 402T microsystem.
For meiotic studies spikelets at appropriate stages were fixed in Carnoy’s fluid for 18–24 h and stored in 70% ethyl alcohol. Acetocarmine smears were utilized for the meiotic study.
In the present study mitotic analysis has been conducted in 88 S. spontaneum clones collected from the North-western states of India, Punjab, and Haryana. The details of the clones, their somatic chromosome numbers, and the frequency of distribution have been given in Table 1 and Fig. 1. The chromosome number determined from well spread mitotic preparations (Fig. 2). Twelve cytotypes with chromosome numbers 2n=40, 48, 50, 52, 54, 55, 56, 60, 64, 70, 72, 74 and 76 have been detected from the area. Among these 2n=40, 48, 56, 64, and 72 can be designated as polyploids with 5x, 6x, 7x, 8x, and 9x respectively, considering x=8 as the basic chromosome number. Clones with chromosome numbers other than these may be aneuploids or the products of intraspecific hybridization taken place at the place of distribution. In this collection the predominant cytotypes observed were 2n=56 (32%). Clones with 2n=54 were in the next position (27%). All the other cytotypes were available in less than 10%, including the 2n=64 cytotype (8%). It has been reported that natural inter and intraspecific hybridizations were responsible for the extensive euploidy and aneuploidy in S. spontaneum (Janaki Ammal 1936, Janaki Ammal and Singh 1936, Raghavan 1953, Bremer 1961, Kandasami 1961, Kandaswamy and Rao 1963, Sreenivasan and Jagathesan 1973).
| Clone name | 2n | Place of collection |
|---|---|---|
| IND 16-1749, IND 16-1752, IND 16-1753, IND 16-1754, IND 16-1758, IND 16-1764, IND 16-1765, IND 16-1766, IND 16-1767, IND 16-1774, IND 16-1781, IND 16-1787, IND 16-1789, IND 16-1796 | 56 | Haryana |
| IND 16-1800 | Chandigarh | |
| IND 16-1802, IND 16-1803, IND 16-1811, IND 16-1814, IND 16-1815, IND 16-1816, IND 16-1818, IND 16-1827, IND 16-1833, IND 16-1834, IND 16-1837, IND 16-1839, IND 16-1843 | Punjab | |
| IND 16-1771, IND 16-1772, IND 16-1775, IND 16-1783, IND 16-1790, IND 16-1793, IND 16-1794, IND 16-1795, IND 16-1797, IND 16-1850 | 54 | Haryana |
| IND 16-1804, IND 16-1805, IND 16-1806, IND 16-1809, IND 16-1817, IND 16-1819, IND 16-1822, IND 16-1824, IND 16-1825, IND 16-1829, IND 16-1831, IND 16-1836, IND 16-1838, IND 16-1849 | Punjab | |
| IND 16-1761, IND 16-1762, IND 16-1778, IND 16-1784, IND 16-1785 | 60 | Haryana |
| IND 16-1844 | Punjab | |
| IND 16-1763, IND 16-1768, IND 16-1769, IND 16-1773, IND 16-1777, IND 16-1782 | 64 | Haryana |
| IND 16-1845 | Punjab | |
| IND 16-1770 | 72 | Haryana |
| IND 16-1808, IND 16-1828, IND 16-1835, IND 16-1847, IND 16-1848 | Punjab | |
| IND 16-1760, IND 16-1786 | 52 | Haryana |
| IND 16-1830, IND 16-1832, IND 16-1842 | Punjab | |
| IND 16-1799 | 70 | Chandigarh |
| IND 16-1826, IND 16-1841 | Punjab | |
| IND 16-1798 | 48 | Haryana |
| IND 16-1801 | Punjab | |
| IND 16-1792 | 50 | Haryana |
| IND 16-1821 | Punjab | |
| IND 16-1759, IND 16-1776 | 74 | Haryana |
| IND 16-1812, IND 16-1813 | 40 | Punjab |
| IND 16-1780 | 76 | Haryana |
Polyploid organisms exist widely in nature. They occur in three main ways: somatic doubling, the formation of non-meiotic gametes, and polyspermy leading to fertilization of the egg by more than one sperm. The production of polyploid organisms by fusion of non-meiotic gametes or by their fusion with meiotic gametes is the main pathway of polyploid formation in plants (Ramsey and Schemske 1998). Panje and Babu (1960) proposed that S. spontaneum cytotypes are distributed mainly in three regions: the first is in Africa and the Mediterranean, with mainly high and medium-high chromosome numbers ranging from 2n=104–128; the second is in the Indian subcontinent including Nepal, eastern and western Pakistan and Ceylon, with mostly low chromosome numbers ranging from 2n=40–80; and the third is in Southeast Asia and the Pacific including Myanmar, with medium and medium-high chromosome numbers ranging from 2n=80–112. In India, different cytotypes are reported in S. spontaneum from different geographical populations. Mehra and Sood (1974) also suggested that the presence of varying chromosome numbers might be due to interspecific hybridization. Wang et al. (1996) and Wen et al. (2001) found that the flowering phases of S. spontaneum with different cytotypes overlapped with each other and that the opportunities for natural hybridization were high enough that many new types of S. spontaneum could be produced.
S. spontaneum shows the highest level of genetic diversity in the Saccharum genus, with nearly 40 different types with varying chromosome numbers (2n=40–128) (Ming et al. 2010, Panje and Babu 1960). Among these categories, chromosome numbers in multiples of eight are more common than multiples of six and ten, suggesting the proposed basic chromosome number of x=8 (Panje and Babu 1960). Cytological evidence from ribosomal DNA sites further confirmed the basic number of x=8 for S. spontaneum (D’Hont et al. 1998). The whole-genome sequence of S. spontaneum (x=8) recently revealed its autopolyploid nature and suggested a process of basic chromosome number reduction from its close relative sorghum (x=10) (Zhang et al. 2018). In the present study also, it has been found that natural intraspecific hybridization between the cytotypes with multiples of 8 resulted in other aneuploid cytotypes which are available in less frequency. All the possible ways of natural hybridization for the evolution of different cytotypes are postulated in Figs. 4 and 5.
In this collection, the lowest chromosome number reported is 2n=40 (5x) and the highest euploidy is 2n=72 (9x). Cytotype with 2n=80 is absent in this collection whereas it was reported earlier from other Northwestern states like Rajasthan and Gujarat (Sobhakumari 2013). Unreduced male and female gamete from different clones is a significant factor in the formation of new polyploids in the natural population Premachandran et al. (2011). While analyzing the existence of 2n=76 cytotype in the collection as the highest reported number we found that there is a possibility for the development of 2n gamete from 2n=40 cytotype to evolve 2n=76 by fusing with n gamete of 2n=72 cytotype. Sreenivasan and Sreenivasan (1994) suggested that 2n=72 cytotypes must have arisen from the natural hybridization involving 2n=80 and 2n=64 cytotypes. In the present study, this possibility has a remote chance as the cytotype with 2n=80 is absent in the population. The role of 2n gamete from 2n=40 cytotype is again assumed in the development of 2n=72 cytotype by fusing with n gamete of 2n=64 cytotype.
The present study suggested that there are different pathways for the evolution of same cytotype. For example, according to earlier reports, 56 chromosome form arose as a hybrid between 2n=48 and 2n=64 forms (Janaki Ammal 1936). But in the present study apart from this pathway, there is a possibility to obtain 2n=56 cytotype by the hybridization between 2n=72 and 2n=40 cytotypes. Yu et al. (2019) indicated that the genetic distances among S. spontaneum with different ploidy levels were different, and hypoploids and hyperploids showed a mesh-type relationship, indicating that the chromosomal evolution of S. spontaneum germplasms might be a mesh-type development rather than in a single direction (Fig. 5). This type of development might provide a better adaptation to different ecological environments.
From our cytological investigation, we found that there are cytotypes with chromosome numbers that are multiples of eight (2n=40, 48, 56, 64, and 72), multiples of ten (2n=40, 50, 60, and 70), and multiples of six (2n=48, 54, 60 and 72). Though many reports are supporting the existence of ploidy in S. spontaneum with basic chromosome number x=8, dissecting out the genome structure and evolutionary relationships among the clones with different ploidy levels are still challenging. Recently Meng et al. (2020) examined a S. spontaneum clone Np-X with 2n=40 chromosomes and they revealed that it was a tetraploid with distinct basic chromosome number x=10. Further studies on population genetic structure and phylogenetic relationship analysis with cytotypes 2n=40 and 2n=64 revealed that there has been a parallel evolution path of genomes for the polyploid series in S. spontaneum with different basic chromosome numbers (Meng et al. 2020).
The production of viable gametes is dependent on the meiosis of the parent. There are chances for the development of clones with different chromosome numbers by meiotic irregularities in naturally grown S. spontaneum (Nair 1972). In the present study, meiosis was done in 10 clones—IND 16-1754, IND 16-1759, IND 16-1960, IND 16-1783, IND 16-1792, IND 16-1801, IND 16-1841, IND 16-1844, IND 16-1845, and IND 16-1847. The majority of clones showed normal meiosis with predominant bivalent formation (Fig. 3A). The meiosis of IND 16-1847 (2n=72) showed the occurrence of univalents at diakinesis and metaphase I. There were occurrences of laggards and bridges which might have resulted in the loss of the chromosomes (Fig. 3B). During the present study multipolar spindle formation and formation of multinuclei cells have been noticed (Fig. 3C, D) This may be responsible for the production of hyper aneuploid gametes as well as hypo aneuploid gametes which will subsequently produce seedling with different chromosome numbers. With these observations in meiosis of the clone IND 16-1847 (2n=72), there is every possibility of getting 2n=70, 72, 74, and 76 cytotypes from this clone itself during inter and intraspecific hybridization.
Mixed-ploidy populations probably act as an effective source of cytogenetic novelty and may facilitate inter-ploidy gene flow. It is a convenient group for the study of factors affecting the origin and establishment of polyploid derivatives. In addition, they offer an opportunity to investigate the evolution of already established cytotypes that may be further subjected to inter-ploidy gene flow and transfer of adaptations (Certner et al. 2017). In the present study altogether five ploidy levels (5x–9x) and several aneuploids were discovered. Among the total cytotype group, aneuploids were present in low frequency (1–7%). According to theoretical predictions, the coexistence of two or more cytotypes within a population is unstable, representing only a transitory state before one of the cytotypes is locally fixed (Levin 1975, Fowler and Levin 1984). Multiple origins of some cytotypes resulted in considerable genetic variation within the population. Recently a genetic diversity analysis of S. spontaneum population revealed that intra-specific variation was significantly higher than interspecific variation in S. spontaneum with different ploidy levels.
S. spontaneum, the most primitive species of sugarcane, has strong environmental adaptability and carries important genetic traits for disease and stress tolerance (Stevenson 1965, Ming et al. 2010). This species is having typical cross-pollinating clones. If different cytotypes of S. spontaneum are pollinated through wind or insects, the fusion of meiotic gametes (n or 2n gametes) occurs, and the resulting species survive under natural selection, S. spontaneum with a new type of chromosome ploidy will exist. In the present study, 88 clones of S. spontaneum collected from Punjab and Haryana were cytologically characterized to find out the evolutionary origin of different ploidy types. It has been found that multiple introgression patterns lead to the origin of the same cytotype grown in the same habitat. When crossbreeding happened among the different ploidy clones of S. spontaneum in the same locality genome shuffling with more genetic complexity will occur and this genome reshuffling will be an additional source of genetic variability in this polyploid population.
The author wishes to thank Dr. Bakshi ram, Director, and Dr. G. Hemaprabha, Head, Crop Improvement Division, ICAR-SBI for the support and facilities provided for the work. She also thanks Dr. Karthikeyan, Principal Scientist, ICAR-SBI, for providing the clones in time to time for the study and Mrs. Remadevi and Dr. Harunipriya for their technical support.
