Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(2): 49-55, 2024 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2670 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Sobhakumari, V.P., & Mohanraj, K. (2024). Application of Genomic In Situ Hybridization (GISH) and tandem repeat sequence ampli- fication for identification of Erianthus – Saccharum introgression. Caryolo- gia 77(2): 49-55. doi: 10.36253/caryolo- gia-2670 Received: April 08, 2024 Accepted: September 24, 2024 Published: November 10, 2024 © 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 Application of Genomic In Situ Hybridization (GISH) and tandem repeat sequence amplification for identification of Erianthus – Saccharum introgression Valiya Purakkal Sobhakumari*, Krishnasamy Mohanraj Crop Improvement Division, ICAR-Sugarcane Breeding institute, Coimbatore-641 007, Tamil Nadu, India *Corresponding author. E-mail: vpsobhakumari@rediffmail.com Abstract. In our experiment a F1 hybrid (GU (04)-28-EO2) obtained from Erianthus procerus (IND 90-776) x Saccharum officinarum (PIO 96-435) was crossed with a com- mercial variety, Co 06027. Resulted BC1 hybrid (GU 12-25) was crossed with a com- mercial cane Co 12009. From this cross ten BC2 progenies were selected and analysed for introgression of Erianthus genome into Saccharum. F1 resulted from 2n+n chro- mosome transmission and was having the whole 40 chromosomes of E. procerus in it. The BC1 and BC2 resulted from n+n transmission. The introgression of E. procerus chromosomes into BC2 ranged from 8-10. Amplification of Erianthus specific tandem repeat (ESTR) sequences was successfully utilized in identification of genuine hybrids of E. procerus x Saccharum. No recombination events between Erianthus X Saccharum could be observed in F1, BC1 and BC2 clones. The current study forms a basis for tar- geted introgression breeding with a different unexploited species of Erianthus, E. pro- cerus in sugarcane improvement programme. Keywords: Erianthus, Saccharum, introgression, Genomic in situ hybridization (GISH), sugarcane, intergeneric hybrid. INTRODUCTION Modern sugarcane cultivars (2n=100-130) are developed from interspe- cific crosses made one century ago among few parent clones of Saccharum officinarum L. (2n=80) the sugar producing species and S. spontaneum L. (2n=40-128), a wild species. Due to interspecific hybridization involving the frequent utilization of a limited number of parental clones the genetic base has become narrow and the cultivars are showing limited resilience to biotic and abiotic stresses. It has been realized that an efficient method to broaden the genetic diversity for increased productivity and better adaptability as well as for providing more disease resistance the responsible genes of wild rela- tives have to be transferred through sugarcane breeding. As one of the most important wild relative of sugarcane, genus Erian- thus has vital role in contribution of desirable characters to sugarcane culti- http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2670 https://doi.org/10.36253/caryologia-2670 https://doi.org/10.36253/caryologia-2670 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 50 Valiya Purakkal Sobhakumari, Krishnasamy Mohanraj vars. Different species of Erianthus have been the focus of several sugarcane breeding programmes as a valuable contributor with many desirable characters like excel- lent vigour with strong root system, high fiber content, good ratooning ability and tolerance to biotic and abi- otic stresses (Ram et al., 2001, Jackson and Hentry, 2011, Fekuhara et al., 2013). In spite of importance of differ- ent species of Erianthus in sugarcane breeding, the major constraint in generating intergeneric hybrids is the cross-incompatibility due to high genetic distance between Saccharum and Erianthus. Another constrain is the difficulty in distinguishing genuine intergener- ic hybrids and self-progeny. The recent development of efficient molecular tools helped in identification of intergeneric hybrids such as PCR based analysis of 5 Sr DNA, SSRs, AFLPs and genomic slot blot hybridization have greatly enabled the identification of intergeneric hybrids Saccharum x Erianthus (D’Hont et al., 1995, Cai et al., 2005, Aitken et al., 2006, Besse et al., 1997) from S. officinarum (female) and Erianthus as male as well as its back crossed variant (D’Hont et al., 1995, Cai et al., 2005, Krishnamurthy et al., 2007, Nair et al., 2006, Piperidis et al., 2000, Piperidis et al., 2010). In order to detect the alien chromosomes and chromosomal seg- ments in a putative hybrid, advanced cytological meth- ods are widely used. Genomic in situ hybridization (GISH is a powerful cytological tool for identifying the introgression status of alien chromosomes in sugarcane (Alix et al., 1998, Jakson and Hentry, 2011). At ICAR-Sugarcane breeding Institute, Coim- batore, India, Erianthus introgression programme has been going on for the last two decades and most of the cases the wild species E. arundinaceus has been used as female parent as a source of potentially valuable traits. In the present study we used a different species of Erian- thus i. e. E. procerus as a female parent to cross with S. officinarum (male parent). Cytologically and also at the molecular level we analysed the F1 and back cross prog- enies of E. procerus x S. officinarum. In sugarcane dur- ing back crosses the problem of shy flowering and non- synchronous flowering makes the breeder to use different sugarcane clones rather than using one of their parents. Such type of crossing methods is recognised as modified back crosses and resulted progenies are referred as back cross progenies. Here we analysed ten BC2 progenies of E. procerus x S. officinarum through Genomic in situ hybridization (GISH) and also analysed the amplification of Erianthus specific tandem repeat sequences to con- firm its hybridity. The genuine hybrid identification and molecular cytogenetic characterization will be helpful for the planning of the breeding strategies for further utiliza- tion of transferred traits from E. procerus. MATERIALS AND METHODS The plant materials used for the study are ten BC2 progenies of a F1 hybrid, GU 04(28) EO2. This F1 hybrid was derived from a cross involving E. procerus (female parent) and S. officinarum (male parent). E. procer- us clone, IND 90-776, was collected from Arunachal Pradesh and S. officinarum clone, PIO 96-435, was an atypical clone derived from interspecific cross undertak- en at the place of origin. The F1 hybrid, GU 04 (28) EO2, was back crossed with a commercial sugarcane cultivar, Co 06027, and obtained BC1 progenies. Among this a confirmed BC1 clone, GU12-25, was crossed with anoth- er commercial variety, Co 12009. From this cross BC2 progenies were raised and analysed for introgression of Erianthus chromosomes. From the BC2 population ten clones were ran- domly selected and its hybridity has been confirmed by amplifying the Erianthus specific tandem repeat (ESTR) sequences (Yang et al., 2019). A PCR reaction mix- ture was prepared (Table 1) and amplification was car- ried out on a Master Cycler (Eppendorf-Nexus gradi- ent) using the primer pairs ESTR-F and ESTR-R (ESTR F: 5’-AGGAAGTTATGGTGGTGGAGTAT-3’; ESTR R: 5’_CGCCATTCCTATTGC-3’). The PCR programme was performed as follows: Pre denaturation at 94°C for 3 min, 34 cycles of 94°C for 1min, 55°C for 35 sec, 72°C foe 30s and 72°C for10 min. PCR products were run in the 1.5% agarose gel electrophoresis Single budded cuttings of E. procerus, S. officinarum, F1, BC1, and BC2cloes were collected from the experi- mental fields of ICAR-Sugarcane breeding institute and planted in the pots. Root tips were collected after 15 days of planting and the somatic chromosome number has been determined according to Sobhakumari and Asmita, 2014. For GISH analysis of BC2 progenies the mitotic chromosome preparations were performed as described by Sobhakumari et al. (2020). The mitotic slides were freeze dried in liquid nitrogen and dehydrated by dip- ping in ethanol. These slides were stored in moisture free Table 1. PCR reaction mixture. Component Volume (µl) Distilled water 15.0 10xPCR buffer 2.0 dNTP mix (10mM) 1.0 ESTR F (primer) 0.5 ESTR R (Primer) 0.5 DNA (50ng/µl) 0.5 Taq enzyme (5U/µl) 0.5 51Genomic in situ hybridization for Erianthus–Saccharum identification slide boxes in room temperature. For GISH analysis the genomic DNA from E. procerus was isolated, fragment- ed to 500-1000bp size and labelled with biotin-16dUTP (thermo Scientific-USA) and used as GISH probe. The methodology followed for GISH analysis was as described previously by Sobhakumari et al., 2021. The hybridization mixture consists of 50 ng of labeled probe of E. procerus, 50% deionized formamide, 10% dextran sulphate, 0.5ng of labeled salmon sperm DNA. After post hybridization washes and FITC incubation the slides were mounted in Vectasheild (Vector labs, UK) mounting medium with DAPI (4,6-diamino 2-phenylindole). GIAH signals were captured using an Axioscope A1 imager f luorescent microscope with Axicam 202 (Carl Zeiss, Gottingen, Germany). Images were processed using Zen 3.0 software (Carl Zeiss, Gottingen, Germany). For each clone 10-15 cells in metaphase were analysed to calculate the number of E. procerus chromosomes. RESULTS AND DISCISSION We reported for the first time the chromosome com- position of fertile E. procerus x S. officinarum F1, BC1 (F1 x sugarcane cultivar, Co 06027), and BC2 (BC1 x sugarcane cultivar, Co 775) hybrids via genomic in situ hybridization (GISH) (Sobhakumari et al., 2020). In this study we utilized a BC1 progeny, GU 12-25 with 20 E. procerus chromosomes to raise BC2 clones. From the BC2 population obtained from GU 12-25 x Co 12009, a set of clones were randomly selected for the classi- cal and molecular cytogenetic analysis. Identifying the genuine hybrids in the intergeneric hybrid population of Erianthus x Saccharum is difficult due to the high selfing rate (Besse et al., 1997). As a preliminary evalu- ation of these BC2 clones for confirmation of its hybrid- ity, PCR amplification was done using Erianthus specific 5S rDNA sequences. The results showed that 5S rDNA sequence amplification was not obtained in some of the genuine hybrids (data not shown). As 5S rDNA has one locus per set of basic chromosomes, it is present only in few chromosomes in the Erianthus genome. Due to unequal segregation and elimination of Erianthus chro- mosomes at different stages, the advanced back cross progenies may not inherit the chromosome that carry the 5S rDNA loci, this may be the reason for not getting amplification of 5S rDNA sequences in BC2 progenies. This showed that Erianthus specific 5S rDNA sequenc- es may not be reliable for the identification of genuine hybrid progenies. Hence the Erianthus specific tandem repeat sequences (ESTR) reported by Yang et al. (2019) was used as a marker to confirm the hybridity of ran- domly selected BC2 progenies. This marker was reported earlier as an E. arundinaceus specific marker and it was showing hybridization sites in the sub telomeric regions at one or both ends of 60 chromosomes of E. arundi- naceus during FISH experiment (Yang et al., 2019). For the first time this marker is used as a hybrid identifica- tion tool in the progenies of E. procerus x Saccharum. In E. procerus parent as well as in its true progenies it has amplified successfully around 380bp (Fig. 1). The ampli- fication was not obtained in the sugarcane varieties. The intergeneric population generated from E. arundinaceus x S. officinarum was initially confirmed with isozyme markers (Deng et al., 2002). However, they could not identify the two hybrids because of absence of banding patterns in different parents. Following this work many reports have come to confirm genuine hybrids with SSR markers, 5S rDNA sequences and internal transcribed spacer (ITS) sequences etc. (Cai et al., 2005a, Cai yet al., 2005b, Zheng et al., 2004). In 2019 Yang et al. reported the AGPR 52/53 sequences for identification of hybrids from Saccharum spp. and E. arundinaceus and the same sequences worked well in our experiments to identify E. procerus x S. officinarum backcross hybrids with modi- fications in the PCR reaction mixture and programme. In recent years many cytogenetic research has been carried out on chromosome transmission of different generations of intergeneric hybrids involving Saccharum and E. arundinaceus (Wu et al., 2014, Huang et al., 2015, Piperidis et al., 2000, 2010, Yang et al., 2019). From these reports it was found that elimination of chromosomes appeared to be a common and non-random event during Figure 1. Electrophoretogram of ten BC2 progenies with their par- ents for amplification of ESTR primer in genomic DNA: 1) 100bp ladder, 2) GU 12-25 (Female parent). 3) Co 12009 (Male par- ent), 4) GU 19-222 5) GU 19-223 6) GU 19-224 7) GU 19-225 8) GU 19-226 9) GU 19-227 10) GU 19-228 11) GU 19-230 12) GU 19-231 13) GU 19-234. 52 Valiya Purakkal Sobhakumari, Krishnasamy Mohanraj intergeneric hybridization in Saccharum. We analysed by genomic in situ hybridization (GISH) the chromosome composition in three generations of E. procerus x Sac- charum intergeneric hybrids: F1 (E. procerus x Sacchar- um), BC1 (F1 x sugarcane cultivar) and BC2 (BC1 x sug- arcane cultivar). Classical cytological studies in ten BC2 clones showed that the 2n number ranged from 88-102 (Fig. 2). In our earlier in situ hybridization studies, it was reported that the F1, GU (04)28 EO2, was showing 2n+n chromosome segregation with 40 chromosomes of E. procerus (Sobhakumari et. al., 2020). BC1, GU 12-25, was contained 20 Erianthus chromosomes with n+n transmission. When GU-12-25 crossed with a commer- cial variety, Co 12009, we could raise BC2 population from which 10 clones were randomly selected for GISH analysis with E. procerus biotin labelled probe. We found that out of 10 BC2 clones studied two clones were with 8 E. procerus chromosomes and only one clone with 9 E. procerus chromosomes whereas the majority of them were with 10 E. procerus chromosomes (7 clones) (Fig. 3). These results revealed that the number of E. procer- us chromosomes in transmission of BC1 to BC2 prog- enies were approximately reduced by half, but we also observed the transmission where reduction was less than half. The details of the parentage, somatic chromosome number and E. procerus introgression pattern in F1, BC1 and BC2 generations of E. procerus x S. officinarum are given in Fig. 4. In earlier reports GISH analysis allows the visualiza- tion of recombination between the species of Saccharum i.e., S. officinarum and S. spontaneum (D’Hont et al., 1996). Different frequencies or abilities of different chro- mosomes to form homologous recombination among S. spontaneum and S. officinarum was reported by Wang et al., in 2021. These species are closely related and they showed interspecific recombination. In spite of that in our study the GISH result from F1, BC1 and BC2 clones did not reveal chromosome exchange between Erianthus and Saccharum chromosomes. The absence of recom- bination may be due to the genetic distance between Erianthus x Saccharum which did not allow the gene transfer by chromosome pairing and chiasma formation Figure 2. Somatic chromosome number of different clones in BC2 generation and its parental clones: a) GU 19-222 (2n=102), b) GU 19-223 (2n=100), c) GU 19-224 (2n=92), d) GU 19-225 (2n= 100), e) GU 19-226 (2n=92), f) GU 19-227 (2n=92), g) GU 19-228 (2n=100), h) GU 19-230 (2n=96), i) GU 19-231 (2n=96), j) GU 19-234 (2n=88), k) GU 12-25 (2n=92) (Female parent l) Co 12009 (2n=104) (Male parent). 53Genomic in situ hybridization for Erianthus–Saccharum identification (Piperidis et al., 2000, 2010). In wheat the role of ‘pairing homeologous 1’ (ph1) gene has been described in pre- venting the pairing between related genera (Hauhar and Chibbar, 1999). In sugarcane no such genes have been reported so far. Chromosome transmission in introgressed popula- tion without inter chromosomal exchange/ recombina- tion revealed that E. procerus genome has been intro- gressed into sugarcane cultivars only by whole chromo- somes. Such chromosomes in the advanced back cross generations are potential source for gene sequencing and SNP marker production after sorting out them sepa- rately. Further back crossing with commercial clones improves the cane traits and juice quality in Erianthus x Saccharum hybrids along with the biotic and abiotic stress tolerance. ACKNOWLEDGEMENTS 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 Figure 3. GISH analysis of BC2 (a-h) clones and its female parent (i): a) GU 19-222, b) GU 19-223, c) GU 19-224, d) GU 19-225, e) GU 19-226, f) GU 19-227, g) GU 19-228, i) GU 19-231, j) GU 12-25. Figure 4. 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