All nonhomologous chromosomes and rearrangements in Saccharum officinarum × Saccharum spontaneum allopolyploids identified by oligo-based painting

Modern sugarcane cultivars (Saccharum spp., 2n = 100~120) are complex polyploids primarily derived from interspecific hybridization between S. officinarum and S. spontaneum. Nobilization is the theory of utilizing wild germplasm in sugarcane breeding, and is the foundation for utilizing S. spontaneum for stress resistance. However, the exact chromosomal transmission remains elusive due to a lack of chromosome-specific markers. Here, we applied chromosome-specific oligonucleotide (oligo)-based probes for identifying chromosomes 1-10 of the F1 hybrids between S. officinarum and S. spontaneum. Then, S. spontaneum-specific repetitive DNA probes were used to distinguish S. spontaneum in these hybrids. This oligo- fluorescence in situ hybridization (FISH) system proved to be an efficient tool for revealing individual chromosomal inheritance during nobilization. We discovered the complete doubling of S. officinarum-derived chromosomes in most F1 hybrids. Notably, we also found defective S. officinarum-derived chromosome doubling in the F1 hybrid Yacheng75-4191, which exhibited 1.5n transmission for all nonhomologous chromosomes. Altogether, these results highlight the presence of variable chromosome transmission in nobilization between S. officinarum and S. spontaneum, including 1.5n + n and 2n + n. These findings provide robust chromosome markers for in-depth studies into the molecular mechanism underlying chromosome doubling during the nobilization, as well as tracing chromosomal inheritance for sugarcane breeding.


Introduction
Sugarcane (Saccharum spp.), a member of the family Poaceae, is a complex heterozygous polyploid plant, exhibiting up to decaploidy or even higher (Grivet and Arruda, 2002;Piperidis and D'Hont, 2020).Sugarcane has been received more attention and is used to produce sugar as well as energy.S. officinarum (2n = 80 or 81, x = 10) exhibits a high sugar content, but poor environmental resistance (Irvine, 1999;Aitken et al., 2006;Piperidis et al., 2010).In contrast, S. spontaneum (2n = 40 128, x = 8, 9, 10), which is distributed widely from the Mediterranean to the Pacific, is highly stress-resistance (Panje and Babu, 1960).S. spontaneum is typically hybridized with S. officinarum (Price, 1957;BT, 1972).Thus, sugarcane is an allopolyploid produced by repeated interspecific hybridization.Allopolyploidy, which results in genetic redundancy, is a source of new variation.In sugarcane, such allopolyploidy results in increased drought tolerance, pest resistance, and biomass production etc. (Osborn et al., 2003;Chen, 2007).Despite its economic importance, the complexity of the sugarcane genetic background has limited classical genetic studies (Barnes and Bester, 2000).The high ploidy level of interspecific Saccharum hybrids, and the variability of the hybridization environment, further complicate sugarcane genetic research.
The complex genetic background of cultivated sugarcane is the result of multiple interspecific hybridization events, particularly between S. officinarum and S. spontaneum.The first artificial interspecific hybrids between these two species were created to overcome disease outbreaks and were followed by repeated backcrossing using S. officinarum as the recurrent female parent to restore high sucrose content.This process, known as 'nobilization', has been central to sugarcane genetic improvement (D'Hont et al., 1996;Ming et al., 2010).Jeswiet has bred a series of excellent parental materials over the past few decades, including the 'POJ' series (Heinz, 1987).This strategy combines the stress resistance of S. spontaneum with the high yield and sugar content of S. officinarum (Jackson, 2005).Grivet et al. found that 15~25% lineages of the sugarcane cultivar R570 were derived from recombination between S. officinarum and S. spontaneum (Grivet et al., 1996).Recent cytogenetic studies have shed light on the basic chromosome number of S. spontaneum and chromosomal inheritance during sugarcane breeding (Cuadrado et al., 2004;Edméet al., 2006).A variety of chromosomal inheritance patterns have been suggested, including "2n + n" and "n + n" (Lalitha and Premachandran, 2007;Paterson et al., 2013).Studies suggest that higher-yielding varieties are the result of "2n+n" heredity (D'Hont et al., 1998).However, the limited chromosomal markers, gDNA probes, and repetitive probes hinders the further exploration of the cytogenetic mechanism underlying sugarcane nobilization.
Fluorescence in situ hybridization (FISH) is being applied in many biological research and has become an indispensable cytogenetic tool (Hu et al., 2014).Through the use of fluorescence, FISH allows the targeting and visualization of specific DNA within whole chromosomes or chromosomal regions (Younis et al., 2015).However, the exact individual chromosome identification is still challenging in plants due to a lack of chromosome-specific markers.Improvements in genomic sequencing have allowed the development of a novel FISH technique called oligonucleotide (oligo)-based FISH (Beliveau et al., 2012;de Oliveira Bustamante et al., 2021).In contrast to conventional FISH, oligo-FISH utilizes bioinformatics-designed probes, which has broadened the scope of cytogenetic researches (Schmutz et al., 2014;Han et al., 2015).Oligo-FISH has been used to study chromosomes and structural genomic variants in several crop species, including potato (Braz et al., 2018), maize (Braz et al., 2021), cucumber (Zhao et al., 2022), and rapeseed (Boideau et al., 2022) etc.Here, we applied oligo-based probes to identify the individual chromosomes of S. officinarum and S. spontaneum, and study the chromosomal heredity of their F 1 hybrids.The results of this study provide a cytological basis for a better understanding of the nobilization in sugarcane breeding.

Plant materials
Plant materials were obtained primarily from Fujian Agriculture and Forestry University and Yunnan Academy of Agricultural Sciences, China.The study materials, including S. officinarum, S. spontaneum, and their F 1 hybrids, are reviewed in Table 1.

Slide preparation
Root tips (~1 cm long) were pretreated with 1,4dichlorobenzene-a-naphthylbromide for 2.5 h, then fixed with freshly-made Carnoy's fixative (ethanol:glacial acetic acid, 3:1) for 24 h at 4 °C.The fixed samples were subsequently rinsed with distilled water and stored in 70% ethanol at -20°C.Root tips were digested with an enzymatic solution (1% pectolase Y-23, 2% pectolyase, 2% cellulase RS, 4% macerozyme R-10) for 3~4 h at 37°C , according to a previously-published protocol (Schwarzacher and Leitch, 1994).The digested root tip suspension was dropped on a slide and then an additional Carnot's fixative was added to disperse the cells.Slide was used for microscopic examination and then the perfect metaphase cells were selected for further oligo-FISH.

Probe labeling and oligo-FISH
All oligo probe sequence libraries are available in Dataset S1 (nph17905-sup-0001-Dataset1.xlsx) (Yu et al., 2022).Oligo probes were synthesized and labeled with either TAMARA-or FAM-, according to the method of Yu et al. (Yu et al., 2022).S. spontaneum-specific repetitive DNA probes (SsRetro1-SsRetro4) were directly-labeled with Cy3-dUTP, according to Huang et al. (Huang et al., 2020).Oligo-FISH was performed according to Braz et al. (Braz et al., 2020), with minor modifications.The treated slides were dehydrated in 75% and 100% ethanol sequentially for each 3 min.Briefly, slides were treated for 1.5 h with pepsin then fixed with 4% formaldehyde for 5 min and with 2× Saline Sodium Citrate (SSC) buffer for 3 min each.The treated slides were sequentially dehydrated with 75% and 100% ethanol for 3 min each.Next, the slides were denatured at 60°C for 90 s with 70% formamide (FD) and rapidly cooled to -20°C.The hybridization solutions (100% FD, 20 × SSC, 50% dextran sulphate (DS) and ~400 ng oligo probes) was added on the center of each slide.Hybridization was carried out at 37°C overnight.Next, the slides were washed using 2 × SSC for 5 min and 10 min, and twice with 1 × PBS for 5 min.The washed slides were then dried and counterstained with the 5 mg/mL 4′,6diamidino-2-phenylindole (DAPI).Chromosome images were captured by Olympus BX53 camera epifluorescence microscope.The contrast of FISH images was adjusted and merged using Photoshop CS6 software.

Unveiling the individual chromosome inheritance of the F 1 hybrids
The 10 chromosome-specific painting probes allow us essentially to identify each nonhomologous chromosomes in S. officinarum and S. spontaneum.Thus, we are interested in knowing how the individual chromosomes inheritance, as well as introgression of the S. spontaneum lineage into S. officinarum.Hence, four F 1 hybrids between S. officinarum and S. spontaneum, Yacheng82-108, Yacheng58-43, Yacheng75-4191 and Yacheng75-409, were selected for chromosomal painting.
We first identified all chromosomes through multiple rounds of oligo-FISH, and then distinguished S. spontaneum-specific chromosomes using S. spontaneum-specific probes applied to the same cell.In Yacheng82-108 (Badila-CN × Yunnan75-2-11), each of the 10 oligo probes generated symmetrical signals through multiple rounds of FISH (Figure 4).The chromosomal colocalization results indicated that Yacheng82-108 contains a total of 112 chromosomes, including 80 derived from S. officinarum and 32 derived from S. spontaneum (Figure 4; Table 2).Each set of nonhomologous chromosomes was uniformly inherited from their parents.
In addition, we chose three other F 1 hybrids from the same male parent Yacheng-spon but with different female S. officinarum parents.For Yacheng58-43, the painting probes generated distinct signals (Figure 5).FISH indicated that Yacheng58-43 (2n) contained 120 somatic chromosomes, including 40 derived from S. spontaneum chromosomes (in which half of each chromosome was derived from the male parent) and 80 derived from S. officinarum.The same chromosomal composition was also found for Yacheng75-409 (Figure 6).Yacheng75-4191 contained 40   chromosomes derived from S. spontaneum (Figure 7, labeled with Arabic numerals), but a different number of chromosomes derived from S. officinarum.Each of the 10 painting probes hybridized to six S. officinarum chromosomes in Yacheng75-4191, suggesting that 1.5n of female parent (S.officinarum) chromosomes were transmitted to Yacheng75-4191 (Figure 7; Table 2).Finally, FISH results indicated that the chromosomal transmission of S. spontaneum is "n" in all F 1 hybrids.

Discussion
Identification of individual chromosome is essential to understand chromosome organization and evolution of related species (Jiang and Gill, 2006;Xiong and Pires, 2011).In plants, chromosome painting with oligo-based probes has been used for karyotyping as well as studies of chromosomal rearrangement, meiotic pairing and recombination (Jiang, 2019).Sugarcane has  one of the most complex genetic backgrounds, and exhibits high degrees of polyploidy as well as aneuploidy (Heinz et al., 1969).The use of chromosome-specific barcodes and painting probes has revealed the complex chromosomal structure and evolutionary history of sugarcane and related species (Meng et al., 2020;Meng et al., 2022;Yu et al., 2022).Sugar-rich S. officinarum typically has either 2n = 80 or 81 chromosomes, with plants containing more than 81 chromosomes likely to be hybrids (Bremer, 1961;Piperidis et al., 2010;Piperidis and D'Hont, 2020).In this study, we found that S. officinarum Fiji and Yuenan niuzhe each possessed eight copies of homologous chromosomes, suggesting that they are typical octaploids (Figure 1).These results support the classical view that S. officinarum is characterized as an autooctaploid with x = 10 (Figure 1).For S. spontaneum, previous cytological evidence indicated that the x = 8 cytotype was derived from x = 10 through consecutive chromosomal breaking and fusion.In this study we used chromosome-specific paints based on the S. officinarum genome and found that both S. spontaneum Yunnan75-2-11 and Yacheng-spon were x = 8.Consistent with previous results, we also found that the S. officinarum 5-like and S. officinarum 8-like chromosomes had been broken and fused.Additionally, we found that SsChr5 is most susceptible to breakage at the S. officinarum-like chromosome 5-6  fusion point in S. spontaneum Yacheng-spon (Figure 2).Notably, this phenomenon is genetically transmitted to the offspring (Figures 5, 6), potentially leading to meiotic instability.
S. spontaneum has been widely used for improving sugarcane genetics due to its excellent agronomic characteristics and the increasing demand for stress-resistant sugarcane cultivars (De Morais et al., 2015;Racedo et al., 2016).In nobilization, the retention of S. spontaneum-derived resistance genes is accompanied by the presence of unreduced S. officinarum gametes, leading to high sugar content (Lu et al., 1994).The varied cytotypes and rich genetic diversity of S. spontaneum have hampered cytogenetic studies in sugarcane (Zhang et al., 2018).Previous studies suggest that either "2n + n" or "n + n" transmission occurs in the F 1 hybrids between S. officinarum and S. spontaneum (Price, 1957;Piperidis et al., 2010).However, the available cytogenetic probes, 5S rDNA, 35S rDNA, and genomic DNA, have been unable to identify all non-homologous chromosomes in these hybrids.Chromosomal inheritance in F 1 hybrids between S. officinarum and S. spontaneum was preliminarily studied based on chromosome numbers (D'Hont et al., 1998;Yu et al., 2018).However, the precise mechanism of individual chromosome transmission remained largely unknown.In this study, we combined chromosome-specific oligo-based probes and S. spontaneum-specific repeat probes to identify all ten sugarcane chromosomes and determine whether they were derived from S. officinarum or S. spontaneum (Figures 4-7).Our system proved highly efficient for tracing the precise chromosomal inheritance pattern in hybrids of S. officinarum and S. spontaneum, and will aid in efforts to further utilize S. spontaneum in sugarcane breeding.
Based on nobilization theory, sugarcane breeders have carried out many researches for improving the genetic background of sugarcane.However, chromosome inheritance mechanism of S. officinarum is still confused and dubious in the hybrids between S. officinarum and S. spontaneum.Previously, "2n + n" chromosome transmission has been found by the method of only using genomic in situ hybridization, but this technique cannot verify whether the S. officinarum chromosomes were fully doubled (Price, 1957;Piperidis et al., 2010;Yu et al., 2018).In this study, by combining S. officinarum-derived chromosome paints and S. spontaneum-specific probes, we that all three studied F 1 hybrids (Yacheng82-108, Yacheng58-43, Yacheng75-409) had "2n + n" chromosome transmission (Figures 4-6).These results suggest that each non-homologous S. officinarum chromosomes was completely doubled.Interestingly, we found one F 1 hybrid (Yacheng75-4191) exhibited "1.5n + n" transmission, suggesting that 1.5 × of S. officinarum chromosomes were inherited (Figure 7).Altogether, these results will broaden our understanding of the sugarcane nobilization between S. officinarum and S. spontaneum.Furthermore, these chromosome-specific identification results will be useful for detailed physical mapping for use in guiding breeders to select suitable parents for sugarcane breeding.

Conclusion
In the present study, the chromosome-specific painting probes derived from S. officinarum can be used for accurately identifying individual chromosome and chromosomal heredity of F 1 hybrids during sugarcane nobilization.We discovered that nonhomologous S. officinarum chromosomes were completely doubling in most F 1 hybrids.However, the F 1 hybrid Yacheng75-4191 exhibited defective chromosome doubling with 1.5n of S. officinarum chromosomes transmission.These results support previous genetic studies of S. spontaneum and provide more useful molecular cytogenetic data for its F 1 hybrids.In addition, these results provide robust chromosome markers for in-depth studies into the molecular mechanism underlying chromosome doubling during the nobilization, as well as tracing chromosomal inheritance for sugarcane breeding.

TABLE 1
The experimental plant materials used in this study.

TABLE 2
Chromosomal composition of F 1 hybrids between S. officinarum and S. spontaneum.