Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(3): 27-36, 2024 Firenze University Press https://riviste.fupress.net/index.php/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2727 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Sobhakumari, V.P., & Mohanraj, K. (2024). Genomic in situ hybridization (GISH) and performance analysis in intergeneric hybrids from five consecutive generations of Eri- anthus x Saccharum. Caryologia 77(3): 27-36. doi: 10.36253/caryologia-2727 Received: Apr 16, 2024 Accepted: Oct 16, 2024 Published: March 25, 2025 © 2024 Author(s). This is an open access, peer-reviewed article pub- lished by Firenze University Press (https://www.fupress.com) and distrib- uted, except where otherwise noted, under the terms of the CC BY 4.0 License for content and CC0 1.0 Uni- versal for metadata. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID VPS: 0000-0003-4165-6826 Genomic in situ hybridization (GISH) and performance analysis in intergeneric hybrids from five consecutive generations of Erianthus x Saccharum V. P. Sobhakumari*, K. Mohanraj Crop Improvement Division, ICAR-Sugarcane Breeding institute, Coimbatore-641 007, Tamil Nadu, India *Correspondeing author. E-mail: vpsobhakumari@rediffmail.com Abstract. Erianthus arundinaceus, one of the species of ‘Saccharum complex’ has a number of important agronomic traits including good ratooning ability, tolerance to both drought and waterlogging, disease resistance and vigor and is of interest as a potential source of parental germplasm to sugarcane breeders. We report here for the first time the chromosome composition, Erianthus chromosome transmission patten and agronomical trait evaluation of Erianthus addition lines in five consecutive gen- erations of E. arundinaceus x Saccharum. The hybridity of randomly selected clones could confirm with Erianthus Specific Tandem Repeat (ESTR) sequences. The results of classical cytology revealed that the mode of transmission of gametes, except in the second generation, followed n+n pattern whereas in second generation (CYM 07-971) it was showing 2n+n with elimination of few chromosomes. Progressive elimination of Erianthus chromosomes is observed in consecutive generations where the F1 showed 30 Erianthus chromosomes and it was ranged from 0-2 in fifth generation. Agronomi- cal trait analysis indicates that further backcrossing with commercial clones with high juice quality or intercrossing among the selected progenies would improve both juice quality and cane traits in E. arundinaceus x Saccharum hybrids. Keywords: Erianthus, Saccharum, introgression, cytology, genomic in situ hybridiza- tion, intergeneric hybrid, sugarcane. Sugarcane belongs to the genus Saccharum and it consists of six spe- cies namely S. officinarum L., S. barberi Jesw., S. sinense Roxb., S. robus- tum Brandes and Jesw. ex Grassl, S. spontaneum L. and S. edule Hassk. The cultivated sugarcane varieties are derivatives of interspecific hybridization between S. officinarum and S. spontaneum. Critical analysis of pedigree of the cultivated varieties revealed that, only a limited number of basic species clones have been contributed to the parental material in sugarcane breeding programmes (Roach, 1989; Hemaprabha et al., 2022). The cytoplasmic vari- ability among major sugarcane varieties under cultivation are not much as only limited S. officinarum clones were used as female parent in early breed- https://riviste.fupress.net/index.php/caryologia https://doi.org/10.36253/caryologia-2727 https://doi.org/10.36253/caryologia-2727 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode https://orcid.org/0000-0003-4165-6826 mailto:vpsobhakumari@rediffmail.com 28 V. P. Sobhakumari, K. Mohanraj ing programmes (Hemaprabha et al., 2022). This may result in vulnerability to disease epidemics and abiotic stresses. In order to widen the cytoplasmic base of sug- arcane cultivars, the wild related species with diverse chloroplast and mitochondrial genomes have been uti- lized in breeding programmes. The “Saccharum com- plex” includes the genera Erianthus Michx, Miscanthus Anderss., Sclerostachya (Hack.) A. Camus and Narenga Bor., besides the Saccharum species and constitute a closely related inter breeding group (Mukherjee 1957; Daniels et al. 1975). The genus Erianthus has established by Michaux in 1803, based on the Greek word ‘Erion’ meaning wool and ‘anthos’ meaning flower, referring to its woolly glumes. It is considered a primitive genus of ‘Saccharum complex’ (Mukherjee 1957). It is wide in distribution occurring in America (New World species), Mediterra- nean, India, China, South East Asia, New Guinea (Old World species). The Old-World species generally placed under section Ripidium, are only important in the evolution and improvement of sugarcane. Among the species of the genus Erianthus sect. ripidium, E. arun- dinaceus (Retz.) Jesw. (2n = 30, 40, 60) distributed in India, China, Indonesia and New Guinea and has many desirable agronomic traits for sugarcane breeding such as disease resistance, drought resistance, high biomass and broad adaptability. Efforts are under way in many research stations to introgress Erianthus germplasm into sugarcane to develop more productive and better adapted sugarcane varieties. Fertile and sterile hybrids were reported from cross- es involving Saccharum and E. arundinaceus (D’ Hont et al. 1995, Besse et al. 1997, Piperidis et al. 2000, 2010, Cai et al. 2005, Wu et al. 2014, Huang et al. 2015) in which either S. officinarum or sugarcane variety used as female and was further back crossed with sugarcane. The major difficulty in transferring the desirable characters from Erianthus into sugarcane is the incompatibility among the genera which prevents further improvement through hybridization. When crosses between two species are not successful due to hybrid sterility and genome elimina- tion bridge crosses were found to be effective in certain cases. The successful use of S. spontaneum as a bridge species for transferring characters from Erianthus to Saccharum was reported by Premachandran et al. 2011. In this case Erianthus has been used as female parent. The production and molecular cytogenetic characteri- zation of intergeneric F1 progeny and back cross prog- enies has been reported earlier (Lekshmi et al. 2016, Premachanran et al. 2017). Later the backcross progenies were developed by crossing with sugarcane commercial varieties to develop near commercial sugarcane clones. Nuclear and cytoplasmic contribution from Erianthus was confirmed in second and third generation hybrids (Lekshmi et al. 2016). Here we aimed to determine the somatic chromo- some number of different backcross progenies of Eri- anthus x Saccharum. We also selected two different sets of BC1, BC2, BC3 and BC4 progenies and used GISH to clarify the pattern of E. arundinaceus chromosome transmission. The aim of the study was to find out the clone that contain 1-2 Erianthus chromosomes which can be further used for sequencing the Erianthus chro- mosomes and also to tag the trait specific Erianthus chromosomes by correlation studies. This work will pro- vide a basis for subsequent genome research as well as trait specific breeding programmes in sugarcane. MATERIALS AND METHODS Plant material The materials used for the study consist of two sets of progenies from BC2, BC3 and BC4 generations. In the first set, the BC 3 progeny, Co 15015, derived from a cross combination of CYM 08-903 and the Sugarcane cultivar Co 94008 (Lekshmi et al. 2016). BC4 progenies (GI 18-1, GI 18-2, GI 18-3, GI 18-4) were from a crossing combination between Co 15015 (female) and sugarcane cultivar, Co 11012 (male). In another set of clones used for the study BC2, BC3 and BC4 progenies were devel- oped by a parallel set of hybridization process. A BC2 clone, CYM 08-922 was selected as female parent and BC3 progeny TWC 82, was derived from a cross combi- nation CYM 08-922 x Sugarcane variety BO91. Four BC4 progenies were developed (FWC 28, FWC 29, FWC 39 and FWC 2) from a cross between TWC 82 x Sugarcane hybrid. The details of the clones used for the study are given in Table 1. To understand the flow of chromosome transmission pattern in successive generations of Erian- thus x Saccharum, the clones from Erianthus (female par- ent), F1 and BC1 were also included in the study. The somatic chromosome number in the F1 and back cross progenies was determined by root tip squash technique (Sobhakumari and Asmita Dutta, 2014). For GISH analysis the mitotic slide preparation was per- formed as per D’Hont et al. 1996 with minor modifica- tions. Single budded cuttings from the hybrids were col- lected at 1.30pm. Excised root tips of about 1 cm were treated with 2mM 8-hydroxy quinolone at room temper- ature for 2h, washed in water and fixed in Ethanol: Ace- tic acid (3:1) for overnight at 4°C. The washed root tips were hydrolyzed in 0.25N HCl and digested at 37°C for 75min in the enzyme solution containing 2.0% cellulase 29Genomic in situ hybridization and performance analysis in Erianthus x Saccharum ONOZUKA R-10 (Himedia) and 20% pectinase (Hime- dia) in citrate buffer. After washing the meristematic tis- sues of the root tips were squashed in a drop of fixative. Cells can be separated by gentle pressing over the cov- erslip with filter paper. Slides were then freezed by dip- ping it in liquid nitrogen. After removing the coverslip, the frozen slide was immediately dehydrated in absolute ethanol and stored in moisture free slide box. Genomic in situ hybridization Total genomic DNA of the Erianthus clone, IK 76-62, was extracted from young leaves using CTAB method (Doyle and Doyle, 1990). Fragmentation (500bp – 1000bp) has been done by sonication. The fragmented DNA of Erianthus labeled with biotin 11-duTP using random primed labeling method as described by manu- facturer (Thermo Scientific, USA) was used as probe. The 30µl hybridization mixture containing 5µlof labeled probe, 15µl deionized formamide, 6 µl 50% dextran sul- phate, 2.25µl 20xSSC, 0.5 µl salmon sperm DNA and 1.25 µl double distilled water was denatured at 100°C for 10min, and then placed immediately in ice. Each chromosomal slide was denatured at 72°Cfor 2min in denaturation solution containing 70% deionized for- mamide and dehydrated in a series of precooled etha- nol solutions (75%, 95%and 100%). After adding 30µl of hybridization solution to the slide it was incubated in a humid box with 2xSSC and at 37°C for overnight. Post hybridization washes in 2xSSC and 50% formamide at 42°C slides were dried. Biotin labeled probe was detected using avidin-FITC (Fluorescein iso thio cynate) (Vector laboratories, Burlingane, CA) by 1h incubation at 37°C. After washing and drying the chronosomes were counter stained with a vectasheild vibrance anti-fading medium with DAPI (4,6 diamino-2-phenylindole) (Vector labo- ratories, Burlingane, CA). GISH signals were captured using a ProgRes Capture Pro image capturing software attached to AxioScope A1fluorescent microscope (Car- lZeiss, Gottingen, Germany). Images were processed using Adobe Photoshop. Screening for drought tolerance and red rot resistance The experimental trial was conducted at East Chithi- rai Chavadi farm of ICAR-Sugarcane breeding institute, Coimbatore, Tamil Nadu, India (110 N, 770E, 427 MSLalti- tude) during 2021-22. The backcross hybrids involving Eri- anthus were planted in split plot desgn, treatments as main plots and genotypes as subplots. The drought treatment was insisted by withholding water during the formative phase of the crop (60-150 DAP). The yield and juice quality data were recorded at 10th month of crop. The drought tolerance indices were calculated as fol- lows: Stress tolerance index (STI) STI = (Yp * Ys) / (Ӯp)2 Yp and Ys are the average yield under normal and moisture stress conditions, respectively. Ӯp is the aver- age yield of all genotypes under normal moisture condi- tions, Screening for red rot resistance under controlled condition testing. The clones were screened for red rot resistance against the virulent inoculum of cf 671 (Colle- totricum falcatum) under controlled condition test- ing (CCT) and the disease reaction was scored as per Mohanraj et al., (1997). RESULTS AND DISCUSSION Chromosome composition of the F1 hybrid GISH analysis of the F1 hybrid CYM 04-420 from the cross between IK 76-78 (Erianthus arundinaceus, 2n=40) x Iritty-2 (Saccharum spontaneum, 2n= 64) revealed that a total of 62 chromosomes (Table 1) of which 30 were from E. arundinaceus and 32 were from S. spontaneum (Fig. 3a), as expected from a classical n+n chromosome transmission (Table. 1) (Lekshmi et al. 2016; Premachandran et al. 2017). The result was con- tradictory to the report of Wu et al. (2014) and Piperidis et al. (2000) where they have reported the presence of anueploids in F1 generation. Figure 1. Erianthus arundinaceus introgression pattern in five gen- erations of Erianthus arundinaceus x Saccharum officinarum. 30 V. P. Sobhakumari, K. Mohanraj Figure 2. Somatic chromosome number of different clones in F1 to BC4 generations: a) IK 76-78 (2n=60), b) Iritty-2 (2n=64), c) CYM 04-420 (2n=62), d) CYM 07-971(2n= 118), e) CYM 08-903 (2n=108), f) CYM 08-922 (2n=108), g) Co 15015 (2n=106), h)TWC 82 (2n=98), i) GI 18-1(2n=108), j) GI 18-2 (2n=109), k)GI 18-3 (2n=107+2F), l) GI 18-4 (2n=108), m) FWC-2 (2n=102), n) FWC-28, (2n=108), o) FWC-29 (2n=102), p) FWC-39 (2n=110). 31Genomic in situ hybridization and performance analysis in Erianthus x Saccharum 2n+ n chromosome transmission in BC1 progeny Due to nonsynchronous flowering of the sugarcane hybrid the fundamental principles of backcross breed- ing are not appropriate in the case of this crop. During intergeneric hybridization in F1 the chromosome inher- ited from divergent parents of different genera are often unable to pair with each other during meiosis which leads to male sterility in F1 hybrid. In order to obtain BC1 generation the F1 hybrid (CYM 04-420) was used as a female parent and a commercial variety Co 775 was used as a male parent. From this cross many BC1 progeny were generated. We considered one BC1 prog- eny, CYM 07-971 for further analysis. The total chromo- some compliment for this hybrid was 2n=118 of which 94 chromosomes were derived from Saccharum and 24 chromosomes were derived from E. arundinaceus (Table 1, Fig. 3b). These results indicated that the BC1 prog- eny was a product of 2n+n transmission. Piperidis et al. (2000), Piperidis et al. (2010) and Wu et al. (2014) were reported similar results. 2n gametes were originated from fusion of two megaspore (Megaspore Tetrad Cell Fusion) or due to chromosome doubling after second meiotic division (Post Meiotic Restitution) (Narayanas- wami (1940), Bremer (1961). n+n chromosome transmission in BC2 and BC3 progeny GISH analysis of two BC2 (CYM 08-903 and CYM 08-922) revealed that these clones were with a total of chromosome compliment of 2n=118 of which 96 chro- mosomes were derived from Saccharum species and 12 chromosomes were from E. arundinaceus (Table 1, Fig. 3 c & d). It was found that the number of E. arundinaceus chromosomes in BC1 parent was 24 and in BC2 proge- ny it was reduced by half. This indicate that BC2 prog- eny (CYM 08-903 and CYM 08-922) were the product of n+n transmission. Parallel back crosses were conducted with these BC2 progenies. i. e., CYM 08-903 x Co 94008 and CYM 08-922 X Bo 91. A nearly commercial cane, Co 15015 with 2n=108 was generated as BC3 progeny from CYM 08-903 x Co 94008. GISH experiment revealed that 102 chromosomes were derived from Saccharum species and 4 from E. arundinaceus. Another BC3 progeny, TWC 82, from the cross CYM 08-922 X BO 91 was with 2n=98 of which 91 chromosomes were from Saccharum and 7 chromosomes were from E. arundinaceus. Our results indicate that the BC3 progeny were the product of n+n transmission. Piperidis et al. (2010, 2013) and Huang et al. (2014) reported that the similar transmission was in BC2 and Bc3 progeny between different species clones of S. officinarum and E. arundinaceus. In this study, both the BC3 progeny, Co 15015 and TWC 82, were derived from BC2 clones CYM 08-903 and CYM 08-922 respectively. Both the BC2 parents were having 12 E. arundinaceus chromosomes. In TWC 82 is seven Erianthus chromosomes were observed. It is found that more than half of the E. arundinaceus chromosomes in CYM 08-922 was transmitted to TWC-82. Where as in the case of Co 15015 only four (less than half) E. arundi- Table 1. Chromosome composition of parents and hybrids of different generations involving Erianthus arundinaceus. S.No Generation Clone Name Total 2n From Saccharum From Erianthus Recombinant No of cells observed 1 Female parent IK76-62 60 - 60 0 10 2 Male parent Iritty-2 64 - - 0 10 3 F1 CYM 04-420 62 32 30 0 15 4 BC1 CYM 07-971 118 94 24 0 12 5 BC2 CYM 08-903 108 96 12 0 16 6 BC2 CYM 08-922 108 96 12 0 15 7 BC3 Co 15015 106 102 4 0 20 8 BC3 TWC 82 98 91 7 0 15 9 BC4 GI 18-1 108 108 - 0 10 10 BC4 GI 18-2 109 107 2 0 15 11 BC4 GI 18-3 108 108 - 0 10 12 BC4 GI 18-4 108 108 - 0 12 13 BC4 FWC-28 108 104 4 0 15 14 BC4 FWC-29 102 99 4 0 15 15 BC4 FWC-39 110 105 5 0 15 16 BC4 FWC-2 102 98 4 0 15 http://S.No 32 V. P. Sobhakumari, K. Mohanraj naceus chromosomes from BC2 (CYM 08-903) was trans- mitted. Though there was difference in the number of E. arundinaceus chromosomes transmitted from female BC2 parents, the transmission pattern was n+n only with addi- tion/deletion of few chromosomes. During the nobilization of S. officinarum X S. spon- taneum 2n+n chromosome segregation happened in the early generations like F1 and BC1. Whereas in the nobilization with E. arundinaceus 2n+n segregation happened during later stages and this slows down the progress of nobilization. In order to develop BC4 prog- enies the BC3 progeny, Co 15015, crossed with another improved Co cane, Co 11012. Cytological analysis of the four BC4 progeny revealed plants with a total chromo- some compliment ranging from 107-109 chromosomes. GISH analysis revealed that out of these four BC4 prog- eny only one clone was having 2 E. arundinaceus chro- mosomes whereas three clones were not having any Erianthus chromosomes. In the parallel back crossing programme TWC 82 (BC3) crossed with an interspecif- ic hybrid and four BC4 progeny were generated. The 2n chromosome number ranged from Saccharum spp. and 3-5 from E. arundinaceus respectively (Fig. 4). Interspecific hybridization or intervarietal hybridi- zation providing frequent utilization of limited number of parental clones resulted in the narrow genetic base of modern sugarcane cultivars and subsequently showing susceptibility to biotic and abiotic stresses. It has become necessary to include wild relatives of Saccharum in breeding programme to broaden the genetic diversity for increased productivity and better adaptability. As one of the most important wild relatives of sugarcane, E. arun- dinaceus has agronomically important genes for sugar- cane breeding. At ICAR-Sugarcane breeding Institute we are regularly utilizing E. arundinaceus in breeding programmes and a series of genuine progeny have been developed in different back crossed generations. Generally, the cytological methods and molecular markers are widely used to specifically detect the alien chromosomes and chromosome segments in the putative back cross progenies. It is found that Genomic in situ hybridization (GISH) is a powerful cytological tool for identifying the introgression pattern of alien chromo- somes in sugarcane background. This study clearly indi- cates the number of back cross generations a breeder has to be developed to incorporate the alien chromosomes. In our study we found that in the BC4 progeny from a cross Co 15015 x Co 11012 only one hybrid was with two Erianthus chromosomes whereas the other proge- nies were without E. arundinaceus chromosomes. Hence Figure 3. Genomic in situ hybridization with E. arundinaceus probe: a) E. arundinaceus x S. spontaneum hybrid, CYM 04-420, with 30 E. arundinaceus chromosomes, b) CYM 07-971 with 24 E. arundinaceus chromosomes, c) CYM 08-903 with 12 E. arundinaceus chromosomes, d) CYM 08-922 with 12 E. arundinaceus chromosomes, e) Co 15015 with four E. arundinaceus chromosomes, f) TWC- 82 with seven E. arundinaceus chromosomes. 33Genomic in situ hybridization and performance analysis in Erianthus x Saccharum this is the stage where the introgression breeding can be stopped as further transmission of Erianthus chromo- somes is not possible in successive generations. Whereas in the parallel cross BC4 progenies from TWC 82 x ISH hybrid 3-5 E. arundinaceus chromosomes were there in which we can go for one more back crossing to get improved clone with minimum Erianthus chromosomes. The agronomic performance of the backcross hybrids involving Erianthus for cane yield quality and red rot resistance is presented in the Table 2. Three hybrids recorded significantly higher cane height than the commercial hybrid Co 86032 (225.0 cm). The sucrose in juice ranged from 8.44% in TWC 82 to 19.98% in Co 15015. Three hybrids recorded significantly higher yield than the commercial check Co 86032 (121.8 t/ha). For red rot resistance, eight were moderately resistant and only one was susceptible. The clones were also screened for water stress during 2021-22. The results showed that the commercial check had a stress tolerance index of 0.761 and five clones recorded significantly higher STI (Table 3). The hybrid TWC 82 recorded the highest STI of 1.678 followed by FWC-2. The entry TWC combined both red rot resistance and water stress tolerance. The juice quality of the hybrids showed significantly lower than commercial check Co 86032 and only one Co 15015 had 19.98% of juice sucrose. Further backcrossing with commercial clones with high juice quality would fur- ther improve both juice quality and cane traits. Similar- ly, Nair et al. 2017 reported backcrossing of E. procerus hybrids with commercial varieties to obtain commer- cially acceptable levels of agronomic traits. Intergeneric hybrid population between Saccha- rum spp. and E. arundinaceus often resulted in false hybrids due to selfing. In order to avoid this, it is highly encouraged to integrate efficient molecular markers with GISH or FISH. Deng et al. (2002) used isozyme mark- ers because of similar banding pattern could not identify the genuine hybrids. Later 5Sr marker used to identify the true hybrid progeny with Erianthus specific 5SrD- NA sequences. It was found that amplification was not obtained beyond BC2 generation. As 5Sr DNA had one locus per set of chromosomes it presents only on a few chromosomes in each genome. Due to unequal segrega- tion of Erianthus chromosomes and also its elimination at different stages the advanced back cross progenies may not inherit the chromosomes that carry the 5SrD- NA loci. Hence Erianthus specific 5Sr DNA sequences may not be reliable for the identification of true hybrid progeny. The Erianthus specific Tandem Repeat sequence (ESTR) reported by Yang et al. (2019) was used as a marker to confirm the hybridity of back cross progeny. In our study true intergeneric hybrids between Saccha- Figure 4. Genomic in situ hybridization with E. arundinaceus probe in 5th generation hybrids: a) GI 18-1 – Nil E. arundinaceus, b) GI 18-2 with two E. arundinaceus chromosomes c) GI 18-3 – Nil E. arundinaceus, d) GI-18-4 – Nil E. arundinaceus, e) FWC-2 with four E. arundi- naceus chromosomes f) FWC-28 with four E. arundinaceus chromosomes g) FWC-29 with three E. arundinaceus chromosomes g) FWC-29 with three E. arundinaceus chromosomes. 34 V. P. Sobhakumari, K. Mohanraj rum spp. and E. arundinaceus could be rapidly identified using PCR with Erianthus Specific Tandem repeat prim- er pair (Fig. 5). It was found that PCR detection results highly coincides with GISH results. Due to the genetic distance between the two genera the chromosome pairing and chiasma formation dur- ing meiosis is not taking place in intergeneric hybrids of Saccharum and Erianthus. From our study it has revealed that E. arundinaceus genome introgressed into Saccharum as whole chromosome by traditional breed- ing. The approaches like QTL mapping and marker assisted breeding in the advanced generations of back crosses (BC3 and BC4) will help to determine the agro- nomic value of individual E. arundinaceus chromosomes. Though E. arundinaceus clones are with many desirable agronomic traits for sugarcane genetic improvement. We have limited knowledge on the complex genome of this hexaploid species. Development and determination of Saccharum – Erianthus introgression lines with one or two E. arundinaceus chromosomes is a necessary step to simplify the genome analysis by dissecting out the alien chromosomes. In this study we identify a clone, GI 18-2, with two Erianthus chromosomes that can be segregat- ed to much lower level in the next generation. In these population identifying genuine hybrid clones with 1-2 E. arundinaceus chromosomes without any recombination or translocation using GISH could be used for dissecting out and sequencing these alien chromosomes. ACKNOWLEDGEMENTS The authors are grateful to the Director, ICAR-Sug- arcane 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. Table 2. Performance of Backcross hybris involving Erianthus for cane yield, juice quality traits and red rot resistance.   Clone Name Cane Ht (cm) Cane dia (cm) SCW (Kgs) Brix (%) Pol (%) Purity (%) CCS % NMC (‘000/ha) Cane yield (t/ha) RR 1 CYM 07-971 210.00 2.61 1.09 14.72 12.06 81.93 8.03 89.00 96.71 MR 2 CYM 08-903 205.00 2.91 1.20 19.07 16.94 88.83 11.74 75.00 89.75 MR 3 CYM 08-922 255.00 2.75 1.38 14.89 12.09 81.20 8.01 101.50 140.07 MS 4 Co 15015 240.00 2.80 1.25 21.63 19.98 92.37 14.10 88.10 110.13 MR 5 TWC 82 265.00 2.95 1.68 11.88 8.44 71.04 5.16 98.30 165.14 MR 6 GI 18-1 220.00 2.46 0.82 19.67 17.60 89.48 12.24 85.00 69.98 MR 7 GI 18-2 235.00 2.57 1.15 15.61 13.17 84.37 8.90 89.00 102.35 MS 8 GI 18-3 235.00 2.71 1.22 19.41 17.11 88.15 11.82 91.50 111.63 MR 9 FWC-28 275.00 2.67 1.15 16.19 13.66 84.37 9.23 94.44 108.61 MS 10 FWC-29 205.00 2.83 1.23 15.47 13.28 85.84 9.05 86.11 105.92 MR 11 FWC-39 255.00 2.51 1.25 15.85 13.48 85.05 9.15 60.19 75.23 MR 12 FWC-2 235.00 2.65 1.45 13.51 10.51 77.79 6.80 100.00 145.00 S   Co 86032 225.00 2.75 1.45 19.91 17.79 89.35 12.37 84.00 121.80 MS CD (P>0.05) 16.23 0.32 0.18 1.12 1.05 4.56 0.98 9.56 11.36 Table 3. Stress tolerance Index (STI) of Backcross hybris involving Erianthus. Sl.No. Clone Cane yield (t/ha) STI Control Stress 1 CYM 07-971 96.71 72.3 0.558 2 CYM 08-903 89.75 68.5 0.490 3 CYM 08-922 140.07 115.5 1.290 4 Co 15015 110.13 75.34 0.662 5 TWC 82 165.14 127.35 1.678 6 GI 18-1 69.98 52.2 0.291 7 FWC-28 108.61 82.3 0.713 8 FWC-29 105.92 78.3 0.662 9 FWC-39 75.23 48.75 0.293 10 FWC-2 145.00 117.35 1.357 11 Co 86032 121.80 78.35 0.761 12 Co 06022 127.41 89.35 0.908 13 CoM 0265 126.00 86.54 0.870 14 Co 775 85.80 37.3 0.255 Overall mean 111.97 80.67 0.771 Treatments (P>0.05) 9.85 Clones (P>0.05) 16.34 0.34 http://Sl.No 35Genomic in situ hybridization and performance analysis in Erianthus x Saccharum REFERENCES Besse P, McIntyre CL, Burner DM, de Almeida CG. 1997. Using genomic slot blot hybridization to assess inter- generic Saccharum x Erianthus hybrids (Andropogon- eae—Saccharinae). Genome 40 (4): 428–432. https:// doi.org/10.1139/g97-057 Bremer G. 1961. 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