The Crop Journal 11 (2023) 1–8

Contents lists available at ScienceDirect

The Crop Journal

journal homepage: www.keaipublishing.com/en/journals/the-crop-journal/

Past and recent advances in sugarcane cytogenetics

Kai Wang a,⇑, Hui Zhang a, Haris Khurshid f, Ayman Esh e, Caiwen Wu d,⇑, Qinnan Wang c,⇑,
Nathalie Piperidis b,⇑
a
School of Life Sciences, Nantong University, Nantong 226019, Jiangsu, China
b
Sugar Research Australia, Mackay 4740, Australia
c
Institute of Nanfan & Seed Industry, Guangdong Academy of Sciences, Guangzhou 510316, Guangdong, China
d
Sugarcane Institute, Yunnan Academy of Agricultural Sciences, Kaiyuan 661600, Yunnan, China
e
Sugar Crops Research Institute, Agriculture Research Center, Giza 12111, Egypt
f
Oilseeds Research Program, National Agricultural Research Centre, Islamabad 44000, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: The Saccharum genus comprises species with large and variable chromosome numbers, leading to chal-
Received 10 May 2022 lenges in genomic studies and breeding improvement. Cytogenetics, including classical and molecular
Revised 20 July 2022 approaches, has played a central role in deciphering the genome structure, classification, and evolution
Accepted 4 August 2022
of the genus Saccharum. The application of fluorescence in situ hybridization using oligonucleotide probes
Available online 2 September 2022
significantly improved our understanding of the complex genomes of Saccharum species. This paper
reviews the application and progress of cytogenetic techniques in Saccharum. Future applications of cyto-
Keywords:
genetics are discussed, as they could benefit both genomic studies and breeding of sugarcane as well as
Cytogenetics
Sugarcane
other plants with complex genomes.
FISH Ó 2022 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by
Chromosome Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY
Oligo-FISH license (http://creativecommons.org/licenses/by/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Brief history and technique development of cytogenetics in sugarcane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. The use of cytogenetics for deciphering the chromosomal constitution of sugarcane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
4. The application of cytogenetics in decrypting the chromosome structure of interspecific and intraspecies hybrids . . . . . . . . . . . . . . . . . . . . . . . . 5
5. Using cytogenetics to dissect the genomic structure of modern sugarcane cultivars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
6. Conclusions and future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Declaration of competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
CRediT authorship contribution statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1. Introduction
Sugarcane (Saccharum spp., Poaceae) is the leading crop used for
sugar production, providing 80% of the world’s sugar, and is an
important biofuel crop used for ethanol and biomass production.
⇑ Corresponding authors.
E-mail addresses: kwang5@ntu.edu.cn (K. Wang), gksky_wcw@163.com (C. Wu),
wangqinnan66@163.com (Q. Wang), NPiperidis@sugarresearch.com.au (N. Piper-
idis).
The Saccharum genus comprises two wild species, i.e., S. robustum
and S. spontaneum, and four formerly cultivated species: S. offici-
narum, S. barberi, S. sinense, and S. edule [1] (Fig. 1). S. officinarum,
also called noble cane [2], is considered to have been domesticated
from S. robustum [3–5] and was the main cultivated sugarcane
worldwide before the end of the 19th century. S. edule might have
originated from S. robustum [6] or could be an interspecific or
intraspecific hybrid between either S. officinarum or S. robustum
and a species in the Saccharum complex [7]. S. sinense and S. barberi
are hybrids of S. officinarum and S. spontaneum [8] and were

https://doi.org/10.1016/j.cj.2022.08.004
2214-5141/Ó 2022 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
K. Wang, H. Zhang, H. Khurshid et al.
Fig. 1. The evolution of species in Saccharum. Evolutionary pathways for the six species and modern cultivars are indicated by lines. Dotted lines indicate the possible
pathways for the formation of S. edule. ‘?’ indicates uncertain parental species of S. edule.
cultivated in different parts of the world long before the origin of
human-made modern cultivars. Modern sugarcane cultivars are
derived from S. officinarum and S. spontaneum hybridization fol-
lowed by repeated backcrossing to S. officinarum, which is also
called nobilization breeding [9].
Saccharum species are all polyploids and have different genome
structures. The genetic complexity of Saccharum species has slo-
wed attempts to understand their genomic structures and has hin-
dered efforts in efficient molecular breeding. Classical and modern
molecular cytogenetics play key roles in understanding the classi-
fication, genome structure and evolution of Saccharum. Here, we
review the progress of cytogenetics in Saccharum by focusing on
the development of molecular cytogenetic techniques and their
utility in this genus, with the anticipation of providing insights
for their application in studies of other species with complex
genomes.
2. Brief history and technique development of cytogenetics in
sugarcane
As early as the 1910s, Barber performed a cytological study in
sugarcane to help establish a classification system [10]. Based on
the observation of chromosome number and morphology, five
groups were classified. Soon after, Bremer reported a series of cyto-
logical studies on chromosome number [11–14] and achieved a
similar grouping. As the number of chromosomes was very high
in all cases studied, Bremer started to investigate chromosome
number using pollen cells at the first mitotic division [11], at which
point chromosomes were paired and reduced to one half in num-
ber. This method was then widely adopted for use in chromosome
number-based cytological studies in sugarcane until the 1960s,
when the mature squash method based on root tips and young
developing leaves was established [15,16].
Classical cytogenetics has played a major role in explaining the
nobilization process in modern cultivar breeding. Prior to the
twentieth century, the global sugarcane industry was extensively
2
The Crop Journal 11 (2023) 1–8
dependent on the noble canes and the canes of India and China.
The varieties were limited in number and yield potential and were
susceptible to diseases and pests. Success in producing interspeci-
fic hybrids between S. officinarum (noble cane) and the wild clone
S. spontaneum represented a breakthrough in sugarcane breeding
because the newly formed progeny demonstrated vigor in both
sugar yield and disease resistance [9]. By chromosome number
investigation, Bremer revealed the phenomenon of 2n + n progeny
from S. officinarum crossed with S. spontaneum [11,12,17] (see
details below). This leads to the nobilized canes having higher
chromosome numbers (100–130) than their parents and therefore
was proposed to be the major driver of increased vigor in sugar
production and adaptability achieved in modern cultivars.
The advent of the molecular cytogenetic technique of fluores-
cence in situ hybridization (FISH) in the 1990s greatly clarified
our understanding of sugarcane chromosome constitution and
structure. D’Hont conducted FISH analyses using 45S and 5S rDNA
probes in S. officinarum and S. spontaneum and revealed basic chro-
mosome numbers of x = 10 for S. officinarum and x = 8 for S. spon-
taneum [18–20]. Soon thereafter, total genomic DNA of S.
officinarum and S. spontaneum was adopted in FISH to decipher
the contribution of the wild species S. spontaneum to modern cul-
tivars [18] and demonstrated that 10%–25% of chromosomes of
modern cultivars were derived from S. spontaneum [18,21–23].
rDNA and genomic DNA FISH assays were also used to study the
origin of the two species S. barberi and S. sinense [8]. However, pro-
gress in molecular cytogenetic studies has severely lagged behind
that in other plants with simple genomes due to the inability to
distinguish individual chromosomes of sugarcane. In fact, the high
levels of repetitive DNA in Saccharum genomes present a substan-
tial challenge in the development of chromosome-specific FISH
probes from the usual bacterial artificial chromosome (BAC) clones
and polymerase chain reaction (PCR) products with exclusively
single- or low-copy sequences [24].
Technical advances in DNA synthesis have allowed for massive
parallel de novo synthesis of thousands of oligonucleotides (oligos).
Recently, these massively synthesized oligos have been success-
K. Wang, H. Zhang, H. Khurshid et al.
fully used to label specific chromosomes or chromosomal regions
by FISH in plants, including sugarcane [25–35]. By applying FISH
using oligo-based chromosome-specific probes, the first accurate
karyotype data were obtained based on the precise identification
of each chromosome [33,34]. Three types of basic chromosome
numbers, x = 8, 9 and 10, were revealed with solid cytogenetic evi-
dence in the wild species S. spontaneum [28,34,35]. By utilizing
chromosome painting oligo probes, the structure and origin of each
chromosome can be deciphered in the complex genome of modern
cultivars [28,35,36]. Therefore, the molecular cytogenetic tech-
nique of oligo-FISH has created a new opportunity for progress in
sugarcane molecular cytogenetics and provides insights for the
characterization of the genomic structure and evolution of the
genus Saccharum.
3. The use of cytogenetics for deciphering the chromosomal
constitution of sugarcane
The chromosome number in a somatic cell (2n) is one of the
most basic pieces of information when describing a species. When
Barber conducted cytogenetic observation of sugarcane in 1916
[10], he realized that the chromosomes of Saccharum species were
numerous but variable among different clones (which were classi-
fied into different groups then). To date, the chromosome numbers
of 2n = 36–170 have been reported from the six Saccharum species
[1,9,28,35,37], demonstrating the high diversity of chromosome
numbers in Saccharum species.
Bremer first claimed a chromosome number of 2n = 80 for S.
officinarum [13]. As a consistent number was further obtained in
subsequent studies, the chromosome number of 2n = 80 was
widely accepted for the species S. officinarum [1,9,38]. Recently,
this chromosome number was further confirmed by FISH using
centromere- and chromosome-specific probes [28,36]. However,
some clones that were identified as S. officinarum by morphology
with a 2n value more or less than 80 were also found. For example,
Jagathesan et al. [38] and Irvine [1] examined 585 and 497 S. offic-
inarum clones, respectively. Among them, 59 and 38 clones were
found with chromosome numbers of 2n – 80, respectively, and
those clones were considered either atypical noble clones or inter-
specific hybrids between S. officinarum and S. spontaneum [1,39].
Although further study is needed to test this hypothesis, cytoge-
netic examination should be applied to identify a S. officinarum
clone with a conceptual karyotype.
It is widely accepted that S. officinarum has an octoploid genome
with the basic chromosome number x = 10. The first cytogenetic
evidence was derived from FISH analyses using rDNA probes, in
which eight homologous signals of 45S rDNA were detected on
eight different chromosomes in several S. officinarum clones
[18,19,40]. Recently, FISH using one set of chromosome-specific
probes further confirmed that the eight signals were localized to
the eight copies of each homologous chromosome, presenting solid
evidence of the basic chromosome number x = 10 for this species
[28,33,34].
S. robustum was discovered in 1928 by Jeswiet during a sugar-
cane collection expedition and was described as a wild cane [41].
S. robustum is considered the ancestral form of S. officinarum, and
the two are closely related in terms of morphology, cytology and
physiology, differing primarily in fiber and sugar contents. Chro-
mosome numbers with a wide range from 60 to 200 have been
proposed for hypothetical S. robustum clones [1,42,43]. However,
after a systematic cytological survey, Price concluded that
S. robustum clones have two major cytotypes, 2n = 60 and
2n = 80, and clones with other chromosome numbers should be
considered hybrids from interspecific or intraspecies crosses [44].
It is plausible to define the clones with 2n = 60 and 2n = 80 as S.
3
The Crop Journal 11 (2023) 1–8
robustum clones if chromosome numbers are used for species iden-
tification. However, the clones with 2n – 60 and 2n – 80 would be
sorted into continuous arrays of new species unless they were
revealed as the results of chromosome rearrangement or inter-
specific hybridization. The basic chromosome number x = 10
was proposed for S. robustum, resulting in hexaploidy and octo-
ploidy for 2n = 60 and 2n = 80 cytotypes, respectively [44]. FISH
using both 45S and 5S rDNA probes also supported this conclu-
sion by revealing six chromosomes harboring the signal for
2n = 60 clones and eight chromosomes for 2n = 80 clones
[19].
For S. spontaneum, at least 31 cytotypes have been identified to
date, displaying the highest levels of diversity among the Saccha-
rum species. Systematic chromosome counts in S. spontaneum were
conducted by Panje and Babu 62 years ago [45]. More than 400
clones collected from India, West Asia, Southeast Asia and Africa
were selected for cytogenetic analysis. A wide range of 2n from
40 to 128 was reported by Panje and Babu [45], covering nearly
all thirty-one 2n cytotypes reported to date [1,46].
The diverse chromosome numbers led to six suggested basic
chromosome numbers, namely, x = 5, 6, 7, 8, 10, and 12 [9]. As
the majority of cytotypes of 2n = 64, 80, and 96 demonstrated a
multiple of 8, the basic chromosome number x = 8 was initially
confirmed [18,19,33]. Phylogenetic analysis revealed that sugar-
cane and sorghum diverged from a common ancestor with the
basic chromosome number x = 10 [47,48]. The reduction in basic
chromosome numbers from 10 to 8 in S. spontaneum has been
shown to involve two chromosome rearrangement events [49].
This was further confirmed by cytological observations in S. spon-
taneum clones [33,34]. However, a question raised here is whether
there were S. spontaneum clones with basic chromosome numbers
of x = 10 and 9 as the ancestors and intermediate stages, respec-
tively. Meng et al. [34] reported the first S. spontaneum clone with
a basic chromosome number of x = 10 by examining a clone with
2n = 40 chromosomes. Soon after, Piperidis and D’Hont reported
two S. spontaneum clones, SES186 (2n = 53) and SES196
(2n = 54), with the same basic chromosome number of x = 9
[28]. Another S. spontaneum clone, 2012–46 (2n = 54), with a basic
chromosome number of x = 9 was also identified by Meng et al.
[35]. The clone 2012–46 was collected from China, and the two
clones SES186 and SES196 were collected from India [45], indicat-
ing a widespread distribution of this cytotype of x = 9 clones. Tak-
ing these cytological findings into account, a two-step process for
the evolution of S. spontaneum from x = 10 to x = 9 to 8 can be pro-
posed (Fig. 2A–C).
The determination of ploidy has always been more problematic
in S. spontaneum clones than in other plants because of the wide
range of chromosome numbers (2n = 40–128) and diverse basic
chromosome numbers proposed (x = 5, 6, 8, 10 or 12). FISH using
rDNA probes has been used to indicate ploidy levels in some clones
of S. spontaneum [18–20,40]. However, the rDNA locus numbers
may vary among S. spontaneum clones with the same ploidy level.
For example, eight 45S rDNA loci were found in the octoploid clone
Yunnan 84–268, but seven were found in another octoploid clone,
SES208 [35], which makes rDNA FISH an unreliable indicator of
ploidy in at least some S. spontaneum clones. Oligo-based probes
provide a powerful tool with which to solve this problem
[28,34,35]. FISH studies based on chromosome-specific oligo
probes identified a total of seven types of ploidies among the 20
S. spontaneum clones studied [34,35], including tetraploidy (4x),
hexaploidy (6x), octoploidy (8x), decaploidy (10x), hendecaploidy
(11x), dodecaploidy (12x) and tridecaploidy (13x) (Fig. 2A–G).
Piperidis and D’Hont [28] also reported S. spontaneum clones with
ploidy levels of 6x, 8x, and 10x. Moreover, they revealed that the
clone Glagah was a tetra-decaploid (14x) (Fig. 2H), which is the
highest-ploidy S. spontaneum clone ever described. In total, eight
K. Wang, H. Zhang, H. Khurshid et al. The Crop Journal 11 (2023) 1–8

Fig. 2. The cytotypes of sugarcane. (A–C) FISH images showing S. spontaneum clones NP-X, 2012–46, and SES208 with basic numbers of x = 10 (A), x = 9 (B), and x = 8 (C),
respectively. CP1, CP2, and CP7, are chromosome painting probes of Chrs. 1, 2, and 7, respectively [35,36]. Ss7 is a chromosome 7-specific probe [34]. Scale bars, 10 lm. (D–H)
FISH images showing S. spontaneum clones Yunnan 82–106 (D), Sichuan 79-I-1 (E), Fujian 89-I-17 (F), Fujian 87-I-4 (G), and Glagah (H) with decaploidy, hendecaploidy,
dodecaploidy, tridecaploidy and tetra-decaploidy, respectively. CP2 and CP7 are chromosome painting probes of Chrs. 2 and 7, respectively. P5, P6, and P9 are chromosome
painting probes of Chrs. 5, 6 and 9, respectively. Scale bars, 10 lm.

4
K. Wang, H. Zhang, H. Khurshid et al.
ploidy types (4x, 6x, 8x, 10x, 11x, 12x, 13x, and 14x) (Fig. 2A–H)
were revealed for S. spontaneum clones.
Limited cytogenetic studies have been conducted for the three
species S. sinense, S. barberi, and S. edule. Only approximately 20
clones with a 2n = 81–120 chromosome number were classified
as S. sinense or S. barberi clones [9], reflecting their interspecific
and intergeneric origins. For the ‘‘edible” cane S. edule, aneuploid
forms with chromosome numbers ranging from 2n = 60–80 were
reported, which is consistent with the hypothesis of an interspeci-
fic or intergenic hybrid origination [50]. Given that all previous
studies were based on classical cytogenetic approaches, further
studies using molecular cytogenetic methods, such as oligo-FISH,
are anticipated to provide more insights into the chromosome con-
stitution and evolution of these three species.
In addition to the cytogenetic assay, flow cytometry was also
conducted to evaluate the DNA content and then to deduce the
chromosome numbers in sugarcane [51–53]. However, to deter-
mine the chromosome number based on flow cytometry, at least
two preconditions are needed: a standard reference clone with a
known chromosome number, ploidy, and DNA content and a reli-
able protocol for sample preparation [54,55]. Chromosome count-
ing based on flow cytometry may be reliable for S. officinarum and
S. spontaneum clones because of their stable basic chromosome
sizes. However, it is a challenge to precisely predict the chromo-
some numbers of modern cultivars because of their hybrid nature
and because they may contain many chromosome rearrangements
[18,22,23,28,56].
4. The application of cytogenetics in decrypting the
chromosome structure of interspecific and intraspecies hybrids
Sugarcane cultivars result mainly from interspecific crosses of
noble canes and the wild species S. spontaneum followed by
repeated backcrossing with noble canes [9,57]. The breeding pro-
gram (also called the nobilization process), which confers the
restoration of noble traits, benefits from the chromosome trans-
mission phenomenon known as female restitution. For female
restitution, the F1 generation of noble cane  S. spontaneum
involves a 2n + n chromosome transmission in which the progeny
has a nonreduced female noble complement (2n) plus the gametic
number (n) of the male. Bremer [11] first reported irregular chro-
mosome transmission when crossing S. officinarum  S. sponta-
neum. Soon after, many studies showed that this is a prevalent
phenomenon in the hybridization of S. officinarum  S. spontaneum
(see reviews by Price [56] and Heinz [9]). This 2n + n transmission
could also be found in the first (BC1) or second (BC2) generations of
backcrosses with S. officinarum, leading to the nobilized canes hav-
ing increased chromosome numbers (100–130), in contrast to their
parents. Intriguingly, this peculiarity is not a consistent feature for
a given interspecific cross between S. officinarum and S. spontaneum
[9,42,58]. Therefore, cytognetic examination may be necessary to
achieve true nobilized interspecific hybidization in sugarcane
breeding.
The mechanism by which S. officinarum transmits its somatic
chromosomes (2n) when pollinated by S. spontaneum remains
uncertain. Bremer [2] suggested that 2n transmission by the female
parent resulted from the formation of egg cells with a doubled
chromosome number through endoduplication after the first mei-
otic division. This hypothesis was postulated because segregation
of the maternal chromosomes was observed among the hybrids
from single crosses of S. officinarum  S. spontaneum, which pre-
cludes the possibility of unreduced egg cell formation. Moreover,
chromosome doubling was observed in the dyad nucleus, support-
ing the hypothesis of endoduplication [17].
5
The Crop Journal 11 (2023) 1–8
No cytogenetic studies on intraspecies hybrids in sugarcane
have been reported. However, the finding of S. spontaneum clones
with odd-number ploidies, such as hendecaploidy (11x) and tride-
caploidy (13x) [34,35], indicates that these clones might be derived
from hybridization between S. spontaneum clones. In fact, it is rea-
sonable to deduce that the decaploid (10x) and dodecaploid clones
(12x) [28,34,35] also originated from intraspecies crosses. For
example, decaploid clones may be derived from hybridization
between tetraploid and hexaploid clones followed by one round
of whole-genome duplication. However, we cannot exclude the
possibility that those clones might have originated from several
rounds of autopolyploidization followed by additional duplication
or deletion events occurring on only one or a few chromosomal
sets. However, this process of duplication or deletion events occur-
ring on only one or a few chromosomal sets is unlikely, as no such
evidence has been reported in plants. Therefore, intraspecies
hybridization may frequently occur within S. spontaneum clones,
which may explain the high level of cytotype diversity in the
clones of S. spontaneum.
5. Using cytogenetics to dissect the genomic structure of
modern sugarcane cultivars
The highly polyploid genomes of both parents, 2n + n transmis-
sion, and high frequency of interspecific recombination led to mod-
ern sugarcane cultivars representing the most genetically complex
crop ever studied. Thus, dissecting the genomic structure of mod-
ern sugarcane cultivars has become one of most pursued interests
of both breeders and genetic researchers who focus on sugarcane.
Recently, whole-genome sequencing has been conducted for the
modern sugarcane cultivar R570 (10 Gb genome size) [49]. A
total of 4660 sugarcane BACs that cover 382 Mb gene-rich regions
of R570 were successfully assembled. However, the assembled
genome accounted for only 3.8% of the putative 10 Gb genome of
R570, reflecting the difficulty in analyzing the genome composition
of sugarcane cultivars using current sequencing techniques.
Using FISH, D’Hont et al. [18] revealed for the first time the con-
tribution of S. officinarum and S. spontaneum to R570. They revealed
that R570 has 2n = 107–115 chromosomes and that approximately
80% of the chromosomes originated from S. officinarum, 10% origi-
nated from S. spontaneum, and an additional 10% had a dual origin.
These results also demonstrated for the first time the occurrence of
interspecific recombination in modern cultivars, renewing the
early knowledge that no recombination occurred between the
chromosomes of S. officinarum and S. spontaneum in modern culti-
vars [9,56]. Subsequent FISH studies in modern sugarcane cultivars
updated the contributions of S. spontaneum to 9% to 23% and chro-
mosomes with interspecific recombination to 5% to 17% [21–
23,28,59].
In addition to FISH using genomic DNA probe, Huang et al. [60]
also reported a new FISH strategy for distinguishing S. spontaneum
chromosomes in modern sugarcane cultivars. By comparing the
repetitive DNA sequences between S. officinarum and S. sponta-
neum, Huang et al. [60] isolated four S. spontaneum-enriched retro-
transposons. The cocktail of probes for four retrotransposons was
used in FISH and generated S. spontaneum-specific painting signals
in the hybrids of S. officinarum and S. spontaneum. Thus, this S.
spontaneum-specific probe was applied in modern sugarcane culti-
vars to distinguish S. spontaneum-derived chromosomes or recom-
bined fragments [60]. Notably, the authors reported that 11.9% to
40.9% of cultivar chromosomes were derived from interspecific
recombination [60], which substantially exceeded the 5%–17% pre-
viously determined by FISH using genomic DNA probes [18,21–
23,59]. This result was attributed to the improved resolution of this
new FISH method [60]. However, this attribution still needs to be
K. Wang, H. Zhang, H. Khurshid et al. The Crop Journal 11 (2023) 1–8

Fig. 3. The toolbox of cytogenetics for Saccharum and other polyploid species. The diagram illustrates the six cytogenetic tools for deciphering the genome structure of
sugarcane or other polyploid species with complex genomes. (A) For chromosome counting, a centromere probe was developed and applied in metaphase FISH in the
octoploid S. spontaneum clone SES208 to facilitate chromosome counting. (B) The species-specific painting probe is developed and used in the FISH assay in the clone YC58-43,
an interspecific hybrid between the S. officinarum clone Badila and the S. spontaneum clone Yacheng. The image shows the FISH result obtained using the S. spontaneum-
specific probe and displayed the distinction of S. spontaneum-derived chromosomes in the hybrid clone YC58-43. (C) The multiple chromosome-specific probes developed
based on regional DNA sequences were hybridized simultaneously in SES208, which allowed us to identify all 64 chromosomes in one cell. (D) Image of FISH mapping using
the chromosomes 2 (Chr. 2) and 8 (Chr. 8) painting probes. In contrast to regional probes (C), chromosome painting probes allow investigation at the whole-chromosome
level. (E) Illustration of the anticipated FISH result obtained using chromosome banding probes in a polyploid cell. To develop the chromosome banding probe, the whole
chromosome sequence is separated into a certain number of segments. These segments are labeled with different colors to allow them to be discriminated from each other.
FISH using a banding probe allows the detection of intrachromosomal rearrangements, such as inversions, duplications and deletions, occurring within a chromosome. (F)
Illustration showing the anticipated FISH result obtained using haplotype probes of chromosome 8 in an autotetraploid cell. In the autotetraploid, each chromosome has four
homologous copies, for example, 8A–8D of chromosome 8. The haplotype panting probe could be designed based on the sequence variation [63] among these four
homologous copies, which will confer the ability to distinguish each homologous copy (8A–8D) of chromosome 8.

confirmed because the cultivars used in the new FISH method were
different from those used for genomic DNA FISH.
Recently, the applications of oligo-based FISH probes have yield
unprecedented insights into the genomic structure of modern sug-
arcane cultivars, especially for the characterization of rearranged
chromosomes [28,34]. Using oligo-based chromosome-painting
probes, Piperidis and D’Hont [28] confirmed the high frequency
of interspecific recombination between the two parental species
S. officinarum and S. spontaneum in modern sugarcane cultivars.
Moreover, translocations within the chromosomes of either S. offic-
inarum or S. spontaneum were also detected. Our study on ten mod-
ern cultivars demonstrated that translocation between S.
spontaneum chromosomes is rare, but translocation between S.
officinarum chromosomes is unexpectedly frequent (an average
of 2.15 translocations per chromosome) in modern cultivars
[36], indicating that pairing between nonhomologous chromo-
somes of S. officinarum might frequently occur during breeding
crosses. Intriguingly, we also observed interspecific recombina-
tion derived from exchange between nonhomologous chromo-
somes of S. spontaneum and S. officinarum. For example, in the
cultivar YT55, an interspecific chromosome was composed of S.
spontaneum Chr. 1 and an unknown S. officinarum chromosome
fragment (non-Chr. 1) [36]. A total of 18 interspecific recombina-
tion events involving nonhomologous chromosomes were
detected from the ten cultivars. However, the frequency of inter-
specific recombination involving nonhomologous chromosomes is
likely underestimated because only four chromosomes were
examined in this study.

6
K. Wang, H. Zhang, H. Khurshid et al.
6. Conclusions and future directions
Molecular cytogenetics, especially the recent development of
oligo-based FISH technology, has played and will continue to play
an important role in revealing the complex genomes of sugarcane.
In fact, there are still many unknowns in sugarcane. One is how the
chromosomes pair and segregate during meiosis. This study will
provide insights into the longstanding question of female restitu-
tion during breeding. Another question is how the individual chro-
mosomes are spatially arranged in sugarcane cells. It is well known
that chromosomes are organized in the nucleus and this spatial
arrangement of the genome plays a crucial role in gene regulation
and genome stability [61,62]. Thus, deciphering chromosome
arrangement will be indispensable for understanding gene expres-
sion regulation in sugarcane.
The recently developed FISH probes for sugarcane, such as the
centromeric probe, species-specific probe, and chromosome-
specific probes covering whole chromosomes or parts of the chro-
mosomes (Fig. 3), have provided the ability to precisely identify
individual chromosomes and reveal basic karyotype information
for the complex sugarcane genome. In fact, precisely counting
chromosomes is still challenging for most sugarcane clones, espe-
cially S. spontaneum clones and modern cultivars. The centromere
repeat-based FISH method may be a preferred way to solve this
problem (Fig. 3A). The chromosome number can be obtained by
readily counting centromere signals with improved accuracy
because centromeric FISH signals are easy to detect and can easily
be distinguished from one another. Moreover, chromosomal frag-
ments that were frequently created during chromosome spread
preparation could be easily distinguished (‘chromosomes’ without
centromere signals in FISH) [36]. Therefore, we believe that further
efforts in the development of cytogenetic tools, such as chromoso-
mal banding or haplotype painting probes (Fig. 3E, F), could reveal
new insights into the genomic structure of sugarcane cultivars and
improve our knowledge of the evolution of sugarcane.
Declaration of competing interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
CRediT authorship contribution statement
Kai Wang: Writing – original draft, Supervision. Hui Zhang:
Writing – original draft. Haris Khurshid: Writing – review & edit-
ing. Ayman Esh: Writing – review & editing. Caiwen Wu: Writing
– review & editing. Qinnan Wang: Writing – review & editing.
Nathalie Piperidis: Writing – review & editing.
Acknowledgments
We would like to thank Zhuang Meng for providing us the FISH
images. This work was supported by the Startup Foundation from
Nantong University (03083074) and the National Natural Science
Foundation of China (32070544).
References
[1] J.E. Irvine, Saccharum species as horticultural classes, Theor. Appl. Genet. 98
(1999) 186–194.
[2] G. Bremer, Problems in breeding and cytology of sugar cane, Euphytica 10
(1961) 59–78.
[3] A. D’Hont, Y.H. Lu, P. Feldmann, J.C. Glaszmann, Cytoplasmic diversity in sugar
cane revealed by heterologous probes, Sugar Cane 1 (1993) 12–25.
7
The Crop Journal 11 (2023) 1–8
[4] Y.H. Lu, A. D’Hont, D.I.T. Walker, P.S. Rao, P. Feldmann, J.C. Glaszmann,
Relationships among ancestral species of sugarcane revealed with RFLP using
single copy maize nuclear probes, Euphytica 78 (1994) 7–18.
[5] S. Schenck, M.W. Crepeau, K.K. Wu, P.H. Moore, Q. Yu, R. Ming, Genetic
diversity and relationships in native Hawaiian Saccharum officinarum
sugarcane, J. Hered. 95 (2004) 327–331.
[6] C.G. Lennoux, Sugarcane collection in New Guinea during 1937, in:
Proceedings of the International Society of Sugar Cane Technologists VI,
Baton Rouge, LA, USA, 1938, pp. 171–182.
[7] J. Daniels, B.T. Roach, Taxonomy and evolution, in: D.J. Heinz (Ed.), Sugarcane
Improvement Through Breeding, Elsevier, Amsterdam, Netherlands, 1987, pp.
7–84.
[8] A. D’Hont, F. Paulet, J.C. Glaszmann, Oligoclonal interspecific origin of ‘North
Indian’ and ‘Chinese’ sugarcanes, Chromosome Res. 10 (2002) 253–262.
[9] D. Heinz, Sugarcane Improvement Through Breeding, Elsevier, Amsterdam, the
Netherlands, 1987.
[10] C.A. Barber, The classification of indigenous Indian canes, Agric. J. India 11
(1916) 371–376.
[11] G. Bremer, A cytological investigation of some species and species hybrids
within the genus Saccharum I, Genetica 5 (1923) 97–148.
[12] G. Bremer, A cytological investigation of some species and species-hybrids of
the genus Saccharum II, Genetica 5 (1923) 273–326.
[13] G. Bremer, The cytology of the sugarcane: a cytological investigation of some
cultivated kinds and their parents, Genetica 6 (1924) 497–525.
[14] G. Bremer, The cytology of the sugarcane, Genetica 7 (1925) 293–322.
[15] T.V. Sreenivasan, Cytogenetic Studies in Saccharum and Allied Genera, Madras
University, India, 1969.
[16] D. Jagathesan, M.J. Ratnambal, Karyotype analysis in Saccharum officinarum,
Nucleus 10 (1967) 159–167.
[17] G. Bremer, Problems in breeding and cytology of sugarcane. IV. The origin of
the increase of chromosome number in species hybrids Saccharum, Euphytica
10 (1961) 325–342.
[18] A. D’Hont, L. Grivet, P. Feldmann, J.C. Glaszmann, S. Rao, N. Berding,
Characterisation of the double genome structure of modern sugarcane
cultivars (Saccharum spp.) by molecular cytogenetics, Mol. Genet. Genomics
250 (1996) 405–413.
[19] A. D’Hont, D. Ison, K. Alix, C. Roux, J.C. Glaszmann, Determination of basic
chromosome numbers in the genus Saccharum by physical mapping of
ribosomal RNA genes, Genome 41 (1998) 221–225.
[20] S. Ha, P.H. Moore, D. Heinz, S. Kato, N. Ohmido, K. Fukui, Quantitative
chromosome map of the polyploid Saccharum spontaneum by multicolor
fluorescence in situ hybridization and imaging methods, Plant Mol. Biol. 39
(1999) 1165–1173.
[21] G. Piperidis, A. D’Hont, D.M. Hogarth, Chromosome composition analysis of
various Saccharum interspecific hybrids by genomic in situ hybridisation
(GISH), in: Proceedings of the XXIV Congress of the International Society of
Sugar Cane Technologists XXIV, Brisbane, Australia, 2001, pp. 565–566.
[22] A. Cuadrado, R. Acevedo, S. Moreno Díaz de la Espina, N. Jouve, C. de la Torre,
Genome remodelling in three modern S. officinarum  S. spontaneum
sugarcane cultivars, J. Exp. Bot. 55 (2004) 847–854.
[23] G. Piperidis, N. Piperidis, A. D’Hont, Molecular cytogenetic investigation of
chromosome composition and transmission in sugarcane, Mol. Genet.
Genomics 284 (2010) 65–73.
[24] J. Jiang, B.S. Gill, Current status and the future of fluorescence in situ
hybridization (FISH) in plant genome research, Genome 49 (2006) 1057–1068.
[25] Y. Han, T. Zhang, P. Thammapichai, Y. Weng, J. Jiang, Chromosome-specific
painting in cucumis species using bulked oligonucleotides, Genetics 200
(2015) 771–779.
[26] F. de Oliveira Bustamante, T.H. do Nascimento, C. Montenegro, S. Dias, L. do
Vale Martins, G.T. Braz, A.M. Benko-Iseppon, J. Jiang, A. Pedrosa-Harand, A.C.
Brasileiro-Vidal, Oligo-FISH barcode in beans: a new chromosome
identification system, Theor. Appl. Genet. 134 (2021) 3675–3686.
[27] L. He, G.T. Braz, G.A. Torres, J. Jiang, Chromosome painting in meiosis reveals
pairing of specific chromosomes in polyploid Solanum species, Chromosoma
127 (2018) 505–513.
[28] N. Piperidis, A. D’Hont, Sugarcane genome architecture decrypted with
chromosome specific oligo probes, Plant J. 6 (2020) 2039–2051.
[29] M. Qu, K. Li, Y. Han, L. Chen, Z. Li, Y. Han, Integrated karyotyping of woodland
strawberry (Fragaria vesca) with oligopaint FISH probes, Cytogenet. Genome
Res. 153 (2017) 158–164.
[30] L. Hou, M. Xu, T. Zhang, Z. Xu, W. Wang, J. Zhang, M. Yu, W. Ji, C. Zhu, Z. Gong,
M. Gu, J. Jiang, H. Yu, Chromosome painting and its applications in cultivated
and wild rice, BMC Plant Biol. 18 (2018) 110.
[31] J. Jiang, Fluorescence in situ hybridization in plants: recent developments and
future applications, Chromosome Res. 27 (2019) 153–165.
[32] Q. Zhao, Y. Meng, P. Wang, X. Qin, C. Cheng, J. Zhou, X. Yu, J. Li, Q. Lou, M. Jahn,
J. Chen, Reconstruction of ancestral karyotype illuminates chromosome
evolution in the genus Cucumis, Plant J. 107 (2021) 1243–1259.
[33] Z. Meng, Z. Zhang, T. Yan, Q. Lin, Y. Wang, W. Huang, Y. Huang, Z. Li, Q. Yu, J.
Wang, K. Wang, Comprehensively characterizing the cytological features of
Saccharum spontaneum by the development of a complete set of chromosome-
specific oligo probes, Front. Plant Sci. 9 (2018) 1624.
[34] Z. Meng, J. Han, Y. Lin, Y. Zhao, Q. Lin, X. Ma, J. Wang, M. Zhang, L. Zhang, Q.
Yang, K. Wang, Characterization of a Saccharum spontaneum with a basic
chromosome number of x = 10 provides new insights on genome evolution in
genus Saccharum, Theor. Appl. Genet. 133 (2020) 187–199.
K. Wang, H. Zhang, H. Khurshid et al.
[35] Z. Meng, Q. Wang, H. Khurshid, G. Raza, J. Han, B. Wang, K. Wang, Chromosome
painting provides insights into the genome structure and evolution of
sugarcane, Front. Plant Sci. 12 (2021) 731664.
[36] K. Wang, H. Cheng, J. Han, A. Esh, J. Liu, Y. Zhang, B. Wang, A comprehensive
molecular cytogenetic analysis of the genome architecture in modern
sugarcane cultivars, Chromosome Res. 30 (2022) 29–41.
[37] G.C. Stevenson, Genetics and Breeding of Sugar Cane, Longmans, Green & Co.,
Ltd., London, UK, 1965.
[38] D. Jagathesan, T. Sreenivasan, M. Ratnambal, Chromosome numbers in noble
sugarcanes, Indian J. Hered. 2 (1970) 89–96.
[39] K.S. Aitken, J.C. Li, P. Jackson, G. Piperidis, C.L. Mcintyre, AFLP analysis of
genetic diversity within Saccharum officinarum and comparison with
sugarcane cultivars, Aust. J. Agric. Res. 57 (2006) 1167–1184.
[40] A. D’Hont, P.S. Rao, P. Feldmann, L. Grivet, N. Islam-Faridi, P. Taylor, J.C.
Glaszmann, Identification and characterisation of sugarcane intergeneric
hybrids, Saccharum officinarum  Erianthus arundinaceus, with molecular
markers and DNA in situ hybridisation, Theor. Appl. Genet. 91 (1995) 320–326.
[41] C.O. Grassl, Saccharum robustum and other wild relatives of ‘‘noble” sugar
canes, J. Arnold Arboretum 27 (1946) 234–252.
[42] S. Price, Cytological studies in Saccharum and allied genera: II. geographic
distribution and chromosome numbers in S. robustum, Cytologia 22 (1957) 40–
52.
[43] T.V. Sreenivasan, J. Sreenivasan, Cytology of Saccharum complex from New
Guinea, Indonesia and India, Caryologia 37 (1984) 351–357.
[44] S. Price, Cytology of Saccharum robustum and related sympatric species and
natural hybrids, in: Technical Bulletin1337, Economic Research Service of the
United States Department of Agriculture, USA, 1965.
[45] R.R. Panje, C.N. Babu, Studies in Saccharum spontaneum distribution and
geographical association of chromosome numbers, Cytologia 25 (1960) 152–
172.
[46] T.V. Sreenivasan, Cytogenetical studies in Saccharum spontaneum, Proc. Indian
Acad. Sci. 81 (1975) 131–144.
[47] N. Jannoo, L. Grivet, N. Chantret, O. Garsmeur, J.C. Glaszmann, P. Arruda, A.
D’Hont, Orthologous comparison in a gene-rich region among grasses reveals
stability in the sugarcane polyploid genome, Plant J. 50 (2007) 574–585.
[48] J. Wang, B. Roe, S. Macmil, Q. Yu, J.E. Murray, H. Tang, C. Chen, F. Najar, G.
Wiley, J. Bowers, M.A. Van Sluys, D.S. Rokhsar, M.E. Hudson, S.P. Moose, A.H.
Paterson, R. Ming, Microcollinearity between autopolyploid sugarcane and
diploid sorghum genomes, BMC Genomics 11 (2010) 261.
[49] O. Garsmeur, G. Droc, R. Antonise, J. Grimwood, B. Potier, K. Aitken, J. Jenkins,
G. Martin, C. Charron, C. Hervouet, L. Costet, N. Yahiaoui, A. Healey, D. Sims, Y.
The Crop Journal 11 (2023) 1–8
Cherukuri, A. Sreedasyam, A. Kilian, A. Chan, M.A. Van Sluys, K. Swaminathan,
C. Town, H. Berges, B. Simmons, J.C. Glaszmann, E. van der Vossen, R. Henry, J.
Schmutz, A. D’Hont, A mosaic monoploid reference sequence for the highly
complex genome of sugarcane, Nat. Commun. 9 (2018) 2638.
[50] B.T. Roach, Chromosome numbers in Saccharum edule, Cytologia 37 (1972)
155–161.
[51] B. Pachakkil, Y. Terajima, N. Ohmido, M. Ebina, S. Irei, H. Hayashi, H. Takagi,
Cytogenetic and agronomic characterization of intergeneric hybrids between
Saccharum spp. hybrid and Erianthus arundinaceus, Sci. Rep. 9 (2019) 1748.
[52] C.J. Metcalfe, J. Li, D. Giorgi, J. Dolezel, N. Piperidis, K.S. Aitken, Flow cytometric
characterisation of the complex polyploid genome of Saccharum officinarum
and modern sugarcane cultivars, Sci. Rep. 9 (2019) 19362.
[53] A.C. Oliveira, M. Pasqual, A.T. Bruzi, L.A. Pio, P.M. Mendonca, J.D. Soares, Flow
cytometry reliability analysis and variations in sugarcane DNA content, Genet.
Mol. Res. 14 (2015) 7172–7183.
[54] J. Dolezel, J. Bartos, Plant DNA flow cytometry and estimation of nuclear
genome size, Ann. Bot. 95 (2005) 99–110.
[55] S.J. Ochatt, Flow cytometry in plant breeding, Cytometry A 73 (2008) 581–598.
[56] S. Price, Cytogenetics of modern sugar canes, Econ. Bot. 17 (1963) 97–106.
[57] B.T. Roach, Cytological studies in Saccharum chromosome transmission in
inter-specific and inter-generic crosses, in: Proceedings of the Thirteenth
Congress of the International Society of Sugar Cane Technologists, Amsterdam,
the Netherlands, 1969, pp. 901–920.
[58] P.A. Kandasami, Interspecific and intergeneric hybrids of Saccharum
spontaneum L. I. functioning of gametes, Cytologia 26 (1961) 117–123.
[59] N. Jannoo, L. Grivet, M. Seguin, F. Paulet, R. Domaingue, P.S. Rao, A. Dookun, A.
D’Hont, J.C. Glaszmann, Molecular investigation of the genetic base of
sugarcane cultivars, Theor. Appl. Genet. 99 (1999) 171–184.
[60] Y. Huang, H. Chen, J. Han, Y. Zhang, S. Ma, G. Yu, Z. Wang, K. Wang, Species-
specific abundant retrotransposons elucidate the genomic composition of
modern sugarcane cultivars, Chromosoma 129 (2020) 45–55.
[61] T. Cremer, C. Cremer, Chromosome territories, nuclear architecture and gene
regulation in mammalian cells, Nat. Rev. Genet. 2 (2001) 292–301.
[62] W.A. Bickmore, B. van Steensel, Genome architecture: domain organization of
interphase chromosomes, Cell 152 (2013) 1270–1284.
[63] L. do Vale Martins, F. Yu, H. Zhao, T. Dennison, N. Lauter, H. Wang, Z. Deng, A.
Thompson, K. Semrau, J.M. Rouillard, J.A. Birchler, J. Jiang, Meiotic crossovers
characterized by haplotype-specific chromosome painting in maize, Nat.
Commun. 10 (2019) 4604.