Abstract
Erianthus is a wild related genus of sugarcane. Its introgression is expected to impart better biomass production, multi ratoonability, and disease and pest resistance to the sugarcane cultivars. In this study, the introgression pattern of the wild genome, E. procerus, has been analyzed in the three consecutive generations. The field performance concerning important agronomic traits and reaction to one of the major diseases, red rot, also has been studied. We report here for the first time the chromosome composition of fertile E. procerus × S. officinarum F1, BC1 (F1×sugarcane cultivar, Co 06027), and BC2 (BC1×sugarcane cultivar, Co 775) hybrids via genomic in situ hybridization (GISH). The F1 resulted from 2n+n chromosome transmission, while BC1 and BC2 from n+n transmission. The hybridity of these clones was confirmed with the presence of both Erianthus and Saccharum specific amplified bands of 5S rDNA. The field evaluation revealed that though BC1 and BC2 hybrids recorded significantly higher stalk number and comparable stalk yields, the juice quality traits need further improvement with few more backcrosses. These Erianthus introgressed hybrid clones proved to be a potential source for red rot resistance in future sugarcane cultivars. This study is aimed to identify the agronomic value of chromosomes from E. procerus and to conduct targeted breeding based on these information.
Sugarcane which belongs to the genus Saccharum in the family Poaceae consists of six species including S. officinarum L., S. spontaneum L., S. robustum E. W. Brandes & Jeswiet ex Grassl, S. barberi Jeswiet, S. sinense Roxb. and S. edule Hassk. The Saccharum together with the four related genera namely Erianthus, Miscanthus, Narenga and Sclerostachya, comprises of the “Saccharum complex” (Amalraj and Balasundaram 2006). Modern sugarcane cultivars are complex interspecific hybrids between the sugar-producing species S. officinarum (2n=80) and the wild species S. spontaneum (2n=40–128). Presently available sugarcane cultivars are derived from intercrossing between the first nobilized hybrids of a few potential clones of S. officinarum and S. spontaneum. While analyzing the pedigree of modern cultivars it has been revealed that only a limited number of cultivated and wild species have contributed towards the parentage of these cultivars (Arceneaux 1965). Hence breeders are concerned about the potentially narrow genetic base of the presently available commercial cultivars of sugarcane and have shown considerable interest in exploiting other germplasm resources to enlarge the genetic base and introduce new genes for yield as well as biotic and abiotic stresses.
Erianthus is a wild relative genus of sugarcane and it has been considered as a valuable genetic resource of the sugarcane cultivars as it is showing genetic divergence with Saccharum. The species of Erianthus has been the focus of several breeding programs as a potentially valuable contributor with many desirable characters like high fiber content, excellent vigor, ratooning ability, and tolerance to abiotic and biotic stresses (Ram et al. 2001, Jackson and Hentry 2011, Fukuhara et al. 2013, Jayabose et al. 2017). Breeders are facing problems in using Erianthus species in sugarcane breeding programs due to the incompatibility that often occurs between Erianthus species and Saccharum and in identifying the genuine hybrids based on the morphological characters. The Erianthus specific molecular markers to overcome the difficulty in identification of intergeneric hybrids were developed (D’Hont et al. 1995, Besse et al. 1996, 1997, Piperidis et al. 2000, Cai et al. 2005). In addition to this GISH was shown to be an effective method for analyzing the genome constitution of the intergeneric hybrids involving Saccharum and Erianthus (D’Hont et al. 1995, Piperidis et al. 2000). At ICAR-Sugarcane Breeding Institute, Coimbatore, Erianthus introgression program has been going on for the last two decades and several hybrids have been developed and characterized (Ram et al. 2001, Nair et al. 2006, Lalitha and Premachandran 2007). In most cases, the E. arundinaceus has been the focus of several breeding programs as a source of potentially valuable traits.
In sugarcane during backcrosses the problem of flowering and synchrony makes the breeder use different sugarcane clones rather than using one of their parents and it is recognized as modified backcross and resultant progenies are referred as BC progenies. In this article, we analyzed the F1 and back cross progenies of intergeneric crosses involving a different species of Erianthus, i.e., E. procerus and Saccharum. Here we report on the genomic characterization and chromosome transmission pattern of Erianthus in three generations of intergeneric hybrids (F1, BC1, and BC2) derived from crosses between E. procerus and Saccharum. As a valuable source for abiotic and biotic stress tolerance, the molecular cytogenetic characterization and agronomic trait evaluation of hybrids involving E. procerus will be helpful for the planning of breeding strategies for further utilization of the traits from E. procerus.
Materials and methods
The plant materials used for the study are F1, BC1, and BC2 of an intergeneric hybrid involving E. procerus (female parent) and S. officinarum (male parent). E. procerus clone, IND 90-776, was collected from Arunachal Pradesh in North East India. PIO 96-435 is an intraspecific hybrid of S. officinarum developed through poly-crossing between typical (2n=80) and atypical (2n=other than 80) clones of S. officinarum. The F1 seedlings were screened morphologically and cytologically. GU 04(28) EO2 was one of the F1 that was confirmed as a genuine hybrid and it was taken for GISH analysis. This hybrid was backcrossed to a commercial sugarcane cultivar Co 06027 and the selected seedlings (BC1 progenies) were subjected to study. Confirmed BC1 hybrid, GU 12-21 was backcrossed with a commercial sugarcane cultivar Co 775, and one BC2 progeny, GU 15-3 was subjected to GISH analysis to understand the parental chromosome segregation pattern. The details of the materials used in the study are described in Fig. 1. In order to confirm the hybridity, F1, BC1, and BC2 were screened with PCR that amplifies the 5S rDNA of Erianthus genome (D’Hont et al. 1995). Amplified products were resolved on 2% agarose gels, stained with ethidium bromide, and documented using a gel documentation system.
The somatic chromosome number of the Erianthus, S. officinarum, F1, BC1, and BC2 clones was determined by root tip squash technique (Sobhakumari and Asmita 2014).
The parents, intergeneric hybrid, BC1, BC2 hybrid along commercial cultivar Co 86032 were evaluated in a randomized block design with a plot size of two rows of 6 m for determining stalk yield and juice quality traits (in 10th month) during 2016–18. Data analysis has been done with the statistical software R 3.3.2 (http://www.R-project.org). The clones were screened for red rot resistance under artificially controlled condition testing against the red rot pathogen cf. 671 (Mohanraj et al. 1997).
Mitotic chromosome preparation for GISH was performed as previously described (D’Hont et al. 1996) with minor modifications. In brief, the hybrids were grown in pots for root tip harvesting. Excised root tips of about 1.0 cm were treated with 2 mM 8-hydroxyquinoline at room temperature for 2 h, rinsed in water, and fixed in ethanol–acetic acid (3 : 1) for about 16 h at 4°C. The root tips were hydrolyzed in 0.25 M HCl and digested in an enzyme solution (2% cellulase Onozuka and 20% pectinase in citrate buffer) at 37°C for 45 min. After washing in distilled water meristematic tissues were squashed in ethanol–acetic acid (3 : 1). Cells can be separated by gentle pressing over the coverslip with a filter paper. Slides were then frozen on dry ice or dipping it in liquid nitrogen. The coverslip was removed and the frozen slide was immediately dehydrated in absolute ethanol and stored in moisture free slide box.
Dried slides were treated with 1 µg µL−1 RNase at 37°C for 45 min and washed 3 times in 2×SSC for 5 min each at room temperature. After drying, the slides were denatured in 70% formamide for 2 min at 72°C and dehydrated in ice-cold ethanol series. Genomic DNA of E. procerus was labeled with biotin-11-dUTP using a random primed labeling method described by the manufacturer (Thermo Scientific, USA) and used as a probe. The hybridization mixture (30 µL per slide) consisted of 50 ng of the labeled probe, 50% deionized formamide, 10% dextran sulfate, 0.5 µL of sheared salmon sperm DNA and 2×SSC. This mixture was denatured for 10 min in a water bath at 100°C and then cooled quickly on the ice. Hybridization was performed overnight in a moist chamber at 37°C. Post hybridization washes were carried out in 2×SSC at 42°C. Biotin-labeled probe was then detected using avidin-FITC by 1 h incubation at 37°C. Post detection washes were carried out in 2×SSC, 1% Triton X-100 in 4×SSC followed by 2×SSC for 5 min at 37°C followed by 5 min incubation in 2×SSC at room temperature. Slides were mounted in Vectasheild (Vector labs, UK) mounting medium with 4,6-diamidino-2-phenylindole. Mitotic cells were analyzed using a Carl Zeiss fluorescence microscope with appropriate filter sets. Images in different filters were captured with Progress Capture Pro software and merged using Adobe Photoshop.
Results
The somatic chromosome number of the parents and F1, BC1 and BC2 progenies of the hybrid involving Saccharum and widely divergent genus Erianthus was determined in root tip mitosis (Fig. 2). The somatic chromosome number of the E. procerus (IND 90-776) and Saccharum (PIO 94-435) parents was 2n=40 and 104 respectively (Fig. 2a, b) and the chromosome number of the F1 hybrid [GU04 (28) EO2] evolved from these parents was 2n=80. The hybrid flowered normally and was utilized for further backcrosses as the female parent and Co 06027 (2n=108) was used as a male parent. Four back cross progenies were subjected to cytological analysis to determine the somatic chromosome number. In these hybrids, the chromosome number ranged from 2n=88–94. One of the confirmed BC1 hybrids, GU 12-21, was again backcrossed with a commercial cultivar Co 775 (2n=110) and the obtained BC2 progeny which showed 2n=103.
The 5S rDNA profiles of intergeneric hybrid, back cross hybrids, and their parents are shown in Fig. 3. The true hybridity has been confirmed by 5S rDNA markers and bands were in the size of approximately 400 bp in Erianthus and 230 bp in Saccharum. The F1 [GU 04(28) EO-20], BC1 (GU 12-21) and BC2 (GU 15-3) had both the bands and confirmed as genuine hybrids of Erianthus.
The agronomic evaluation of F1, BC1, and BC2 along with their parents and the commercial cultivar Co 86032 for cane yield and juice quality traits are given in Table 1. The F1 hybrid GU 04(28) EO-2 recorded the highest stalk number 151,250 ha−1) followed by GU 15-3 (127,140) and GU 12-21 (121,140). The intergeneric hybrid recorded the lowest stalk diameter (1.26 cm), single stalk weight (0.65 kg), and juice quality traits compared to BC hybrids and the commercial clones. The F1 hybrid and BC1 were red rot-resistant and BC2 was moderately resistant under controlled condition testing against the highly virulent strain cf. 671.
Table 1. Performance of intergeneric and backcross hybrids for stalk yield, juice quality traits, and red rot resistance.
| Clone | NMS* (ha−1) | Stalk height (cm) | Stalk diameter (cm) | SSW** (kg) | Juice brix (%) | Sucrose (%) | CCS (%) | Purity (%) | Stalk yield (t ha−1) | Red rot rating |
|---|
| GU 04(28) EO2 | 151.25 | 235.33 | 1.26 | 0.65 | 12.51 | 7.88 | 4.40 | 62.99 | 98.31 | R |
| PIO 96-435 | 70.45 | 190.10 | 2.45 | 0.98 | 19.10 | 16.28 | 11.06 | 85.24 | 69.04 | S |
| GU 12-21 | 121.14 | 230.50 | 2.10 | 0.90 | 17.56 | 14.18 | 9.36 | 80.75 | 109.02 | R |
| GU 15-3 | 127.60 | 245.60 | 2.34 | 1.01 | 18.25 | 14.90 | 9.90 | 81.64 | 128.87 | MR |
| Co 06027 | 88.89 | 260.35 | 2.92 | 1.33 | 19.26 | 17.31 | 12.07 | 89.88 | 118.22 | MR |
| Co 775 | 57.78 | 200.50 | 3.05 | 1.14 | 18.24 | 16.10 | 11.13 | 88.27 | 65.87 | MS |
| Co 86032 | 102.22 | 240.42 | 2.78 | 1.08 | 20.67 | 18.69 | 13.07 | 90.42 | 110.39 | MS |
| CD (0.05) | 15.01 | 18.16 | 0.45 | 0.18 | 1.98 | 1.84 | 1.73 | 6.18 | 16.24 | |
*Number of millable stalks, **single stalk weight
The GISH analysis of the F1 hybrid, GU 04(28) EO2 revealed that out of 80 chromosomes, 40 chromosomes were from E. procerus and 40 chromosomes from S. officinarum (Fig. 4a). Among the BC1 progenies GU 12-21 (2n=94) was subjected to GISH analysis with the biotin labeled genome DNA of E. procerus revealed that out of 94 chromosomes, 20 chromosomes were derived from E. procerus (Fig. 4b). In the next generation, the BC2 (GU 15-3) displayed 2n=103, of which 10 chromosomes were derived from Erianthus species (Fig. 4c).
Discussion
In recent years, sugarcane breeders are giving more attention towards the use of Erianthus spp. in several sugarcane breeding programs as a potential source for valuable traits like high biomass, excellent vigor, multi ratoonability, disease, and pest resistance and tolerance to environmental stresses (Piperidis et al. 2000, Jackson and Hentry 2011). Due to high genetic diversity between the two genera, limited success could only be achieved in the breeding programs because of low compatibility. Difficulties are also faced to identify the genuine progeny produced from the cross between these two genera on account of a large proportion of selfed progeny and identification of hybrids based on morphological characters is very challenging. Molecular markers had been utilized for the identification of true hybrids involving Saccharum × Erianthus (Besse et al. 1996, 1997, Alix et al. 1999, Nair et al. 2006, Piperidis et al. 2000, Cai et al. 2005). Particularly 5S rDNA markers are being successfully used in the identification of Erianthus hybrids (D’Hont et al. 1995, Fukuhara et al. 2013, Pachakkil et al. 2019). In our study also the F1, BC1, and BC2 hybrids of Erianthus were undoubtedly confirmed as genuine hybrids using 5S rDNA markers. Though the hybridity could be confirmed in intergeneric hybrids with markers, the presence of alien chromosomes, their number, and possible recombinant chromosomes could be confirmed only through GISH techniques (D’Hont et al. 1995, Piperidis et al. 2000, 2010a).
The classical cytological techniques revealed the somatic chromosome number of the F1 hybrid, GU 04(28) EO-2 as 2n=80. The expected exact chromosome number from n+n transmission is 2n=72 (20+52). In our studies, the confusion regarding chromosome transmission has been cleared with GISH. We observed that the F1 hybrid showed 2n+n transmission with the elimination of 12 chromosomes from Saccharum parent. The hybrid showed 40 chromosomes of E. procerus on using E. procerus genome DNA as the probe during GISH has been suggested that when crosses involved distantly related species there is a chance for 2n+n chromosome transmission as in the case of S. officinarum × S. spontaneum crosses (Bremer 1923, 1961, Parthasarathy 1948).
The male parent, PIO 96-435, is an intraspecific hybrid developed by intercrossing typical and atypical clones of S. officinarum in the germplasm. An atypical S. officinarum would have been originated by natural hybridization with related species at its place of origin. Hence there is a chance for S. spontaneum genome in it and its presence has been proved through molecular cytogenetic tools (Piperidis et al. 2010a, Yu et al. 2018). This genome might have induced the 2n chromosome transmission from the other parent (E. procerus). In crosses involving Saccharum and E. arundinaceus n+n transmission had been generally reported in F1 hybrids in which Saccharum spp. was used as the female parent and Erianthus as the male parent (Janaki Ammal 1941, Rao et al. 1963, Kandasami 1964, D’Hont et al. 1995, Piperidis et al. 2000, 2010b, Wu et al. 2014). This is the first report in which Erianthus was used as the female parent and its 2n gamete transmission in F1 has been proved by GISH.
The high rate of aneuploidy in F1 hybrid, due to the elimination of 12 chromosomes contributes to the production of unbalance gametes resulting in a high degree of pollen sterility in the F1 hybrid. o obtain back cross progenies, the F1 hybrid, GU 04(28) EO-2, was used as the female parent, and the cultivar Co 06027 (2n=108) used as the male parent. Mitotic studies revealed the somatic chromosome number of four BC1 progenies ranging from 2n=88–94 with n+n chromosome segregation. Among these, the clone with the highest chromosome number, GU 12-21, was subjected to GISH analysis to understand the transmission pattern of alien chromosomes from E. procerus. Twenty chromosomes of E. procerus could be revealed in this BC1 hybrid. This BC1 progeny was later crossed with a cultivar Co 775 (2n=110) and root tip mitotic studies revealed that in BC2 the somatic cell chromosome number as 2n=103. GISH analysis revealed the n+n segregation pattern with chromosomes of E. procerus.
In three consecutive generations, the GISH confirmed that there were no recombination events between Saccharum and E. procerus chromosomes. This is consistent with the earlier reports (Piperidis et al. 2000, 2010b, Lekshmi et al. 2017, Premachandran et al. 2017). As these genomes are distantly related it is suggested that E. procerus may be introgressed into cultivars as only in whole chromosomes by conventional breeding programs.
The F1 hybrid, GU 04(28) EO-2, having Erianthus cytoplasm and whole-genome (2n) of Erianthus with S. officinarum genome is a potential source for diversifying the cytoplasmic and genomic base of the cultivars to be developed in the future. It is proven that in the F1 hybrid, 2n+n transmission has occurred with chromosome elimination from S. officinarum to overcome a genetic imbalance that occurred due to the presence of two diverged genomes in the hybrid. In the earlier reports (Piperidis 2010b, Wu et al. 2014) where Saccharum was using as the female parent and Erianthus as the male parent, the F1 showed n+n transmission. In contradictory to these reports in our study with Erianthus as a female parent F1 was showing 2n+n transmission and BC1 was showing n+n transmission. In both these cases at the stage of BC1, half the genome of the Erianthus is retaining. In our study either BC1 or BC2 progenies were not showing 2n+n chromosome transmission and this might be to avoid genomic imbalance by increasing the Erianthus components in the genome.
The F1, BC1, and BC2 recorded significantly higher stalk number compared to commercial cultivar Co 86032. Though the backcross hybrid GU 15-3 recorded higher stalk yield but had significantly lower juice Brix%, Sucrose %, CCS%, and Purity% than the commercial cultivars which indicates further improvement is required for improving juice quality. There was an improvement in BC2 for single stalk weight and juice quality over BC1 and F1. For stalk yield also, BC2 had shown an improvement of 31.02% over F1 and 18.16% over BC1. There was a not significant difference between BC1 and BC2 for juice quality, however, BC2 recorded a 60.27% improvement over F1 hybrid and 5.02% over BC1. The F1 and BC hybrids were resistant to red rot which is the most devastating disease in both tropics and subtropics of the Indian subcontinent. The Erianthus introgressed clones proved to be a potential source for diversifying red rot-resistance in sugarcane. The selection and utilization of promising back cross progenies in future hybridization programs will develop sugarcane cultivars with a broad genetic base and adaptability with better productivity.
Acknowledgments
The authors are grateful to the Director, ICAR-Sugarcane Breeding Institute, Coimbatore, India, for the encouragement and providing facilities to conduct the study. The authors also acknowledge the funding support of the project (EEQ/2019/000124) awarded by the Science and Engineering Research Board (SERB), Department of Science and Technology, New Delhi. India.
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