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)13,14. The result was contradictory to the report of Wu et al. and Piperidis et al. 10,7 where they have reported the presence of anueploids in F1 generation.
2n + n chromosome transmission in BC1 progeny:
Due to nonsynchronous flowering of the sugarcane hybrid the fundamental principles of backcross breeding are not appropriate in the case of this crop. During intergeneric hybridization in F1 the chromosome inherited 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 progeny, CYM 07-971 for further analysis. The total chromosome 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 progeny was a product of 2n + n transmission as earlier reports7,8,10. 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) 19,20.
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 chromosomes were derived from Saccharum species and 12 chromosomes were from E. arundinaceus (Table 1, Fig. 3c & d). It was found that the number of E. arundinaceus chromosomes in BC1 parent was 24 and in BC2 progeny it was reduced by half. This indicate that BC2 progeny (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. Earlier the similar transmission pattern was observed in BC2 and BC3 progeny between different species clones of S. officinarum and E. arundinaceus 8,11,21.
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. arundinaceus chromosomes from BC2 (CYM 08-903) was transmitted. Though there was difference in the number of E. arundinaceus chromosomes transmitted from female BC2 parents, the transmission pattern was n + n only with addition/deletion of few chromosomes.
During the nobilization of S. officinarum X S. spontaneum 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 progenies 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 chromosome compliment ranging from 107–109 chromosomes. GISH analysis revealed that out of these four BC4 progeny only one clone was having 2 E. arundinaceus chromosomes whereas three clones were not having any Erianthus chromosomes. In the parallel back crossing programme TWC 82 (BC3) crossed with an interspecific 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 hybridization 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. arundinaceus has agronomically important genes for sugarcane 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 chromosomes in sugarcane background. This study clearly indicates 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 progenies were without E. arundinaceus chromosomes. Hence this is the stage where the introgression breeding can be stopped as further transmission of Erianthus chromosomes 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 further improve both juice quality and cane traits. Similar research on backcrossing of E. procerus hybrids with commercial varieties to obtain commercially acceptable levels of agronomic traits was reported in 201722.
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 |
Intergeneric hybrid population between Saccharum 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. Later used isozyme markers because of similar banding pattern could not identify the genuine hybrids23. Earlier we used 5Sr marker to identify the true hybrid progeny with Erianthus specific 5SrDNA 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 segregation of Erianthus chromosomes and also its elimination at different stages the advanced back cross progenies may not inherit the chromosomes that carry the 5SrDNA 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)24 was used as a marker to confirm the hybridity of back cross progeny. In our study true intergeneric hybrids between Saccharum spp. and E. arundinaceus could be rapidly identified using PCR with Erianthus Specific Tandem repeat primer 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 during 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 breeding. The approaches like QTL mapping and marker assisted breeding in the advanced generations of back crosses (BC3 and BC4) will help to determine the agronomic 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 segregated 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.