Ki Wng,Hui Zhng,Hris Khurshid,Aymn Esh,Ciwn Wu,Qinnn Wng,Nthli Pipridis

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

Keywords:Cytogenetics Sugarcane FISH Chromosome Oligo-FISH

ABSTRACT The Saccharum genus comprises species with large and variable chromosome numbers,leading to challenges in genomic studies and breeding improvement.Cytogenetics,including classical and molecular approaches,has played a central role in deciphering the genome structure,classification,and evolution of the genus Saccharum.The application of fluorescence in situ hybridization using oligonucleotide probes 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 cytogenetics are discussed,as they could benefit both genomic studies and breeding of sugarcane as well as other plants with complex genomes.

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.The Saccharum genus comprises two wild species,i.e.,S.robustum and S.spontaneum,and four formerly cultivated species:S.officinarum,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 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 followed by repeated backcrossing to S.officinarum,which is also called nobilization breeding[9].

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.

Saccharum species are all polyploids and have different genome structures.The genetic complexity of Saccharum species has slowed attempts to understand their genomic structures and has hindered efforts in efficient molecular breeding.Classical and modern molecular cytogenetics play key roles in understanding the classification,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 cytological 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 number.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 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 interspecific 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 fluorescence 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 chromosome numbers of x=10 for S.officinarum and x=8 for S.spontaneum[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 cultivars[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,progress 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 substantial 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 successfully 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 evidence 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 technique 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 classified 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.officinarum 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 interspecific hybrids between S.officinarum and S.spontaneum[1,39].Although further study is needed to test this hypothesis,cytogenetic 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 sugarcane 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.Chromosome 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.robustum clones if chromosome numbers are used for species identification.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 interspecific hybridization.The basic chromosome number x=10 was proposed for S.robustum,resulting in hexaploidy and octoploidy for 2n=60 and 2n=80 cytotypes,respectively[44].FISH using both 45S and 5S rDNA probes also supported this conclusion 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 Saccharum 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 sugarcane 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.spontaneum 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,respectively.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],indicating a widespread distribution of this cytotype of x=9 clones.Taking 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 proposed(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 ploidy types(4x,6x,8x,10x,11x,12x,13x,and 14x)(Fig.2A–H)were revealed for S.spontaneum clones.

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 μm.(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 μm.

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 interspecific 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 constitution 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 determine 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 reliable protocol for sample preparation[54,55].Chromosome counting 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 chromosome 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 program(also called the nobilization process),which confers the restoration of noble traits,benefits from the chromosome transmission phenomenon known as female restitution.For female restitution,the F1generation 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 chromosome transmission when crossing S.officinarum×S.spontaneum.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 having 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 meiotic 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 precludes the possibility of unreduced egg cell formation.Moreover,chromosome doubling was observed in the dyad nucleus,supporting the hypothesis of endoduplication[17].

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 tridecaploidy(13x)[34,35],indicates that these clones might be derived from hybridization between S.spontaneum clones.In fact,it is reasonable 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 occurring 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 transmission,and high frequency of interspecific recombination led to modern sugarcane cultivars representing the most genetically complex crop ever studied.Thus,dissecting the genomic structure of modern 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 contribution 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% originated 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 cultivars[9,56].Subsequent FISH studies in modern sugarcane cultivars updated the contributions of S.spontaneum to 9%to 23%and chromosomes 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.spontaneum,Huang et al.[60]isolated four S.spontaneum-enriched retrotransposons.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 cultivars to distinguish S.spontaneum-derived chromosomes or recombined 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%previously 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 confirmed because the cultivars used in the new FISH method were different from those used for genomic DNA FISH.

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.spontaneumspecific 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.

Recently,the applications of oligo-based FISH probes have yield unprecedented insights into the genomic structure of modern sugarcane 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.officinarum or S.spontaneum were also detected.Our study on ten modern 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 chromosomes of S.officinarum might frequently occur during breeding crosses.Intriguingly,we also observed interspecific recombination derived from exchange between nonhomologous chromosomes 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 recombination events involving nonhomologous chromosomes were detected from the ten cultivars.However,the frequency of interspecific recombination involving nonhomologous chromosomes is likely underestimated because only four chromosomes were examined in this study.

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 restitution during breeding.Another question is how the individual chromosomes 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 expression regulation in sugarcane.

The recently developed FISH probes for sugarcane,such as the centromeric probe,species-specific probe,and chromosomespecific probes covering whole chromosomes or parts of the chromosomes(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,especially 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 fragments 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 chromosomal 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 financial 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 & editing.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).