Meiotic abnormalities in sugarcane (Saccharum spp.) and parental species: Evidence for peri- and paracentric inversions

The modern cultivars of sugarcane (Saccharum spp.) are highly polyploid and accumulate aneuploidies due to their history of domestication, genetic improvement and interspecific hybrid origin involving the domesticated sweet species Saccharum officinarum (‘noble cane’) and the wild Saccharum spontaneum, both with an evolutionary history of polyploidy. The first hybrids were backcrossed with S. officinarum, and selection from progenies in subsequent generations established the genetic basis of modern cultivars. Saccharum genome complexity has inspired several molecular studies that have elucidated aspects of sugarcane genome constitution, architecture and cytogenetics. Herein, we conducted a comparative analysis of the meiotic behaviour of representatives of the parentals S. officinarum and S. spontaneum, and the commercial variety, SP80-3280. S. officinarum, an octoploid species, exhibited regular meiotic behaviour. In contrast, S. spontaneum and SP80-3280 exhibited several abnormalities from metaphase I to the end of division. We reported and typified, for the first time, the occurrence of peri- and paracentric inversions. Using in-situ hybridisation techniques, we were able to determine how pairing association occurred at diakinesis, the origin of lagging chromosomes and, in particular, the mitotic chromosome composition of SP80-3280. Interestingly, S. spontaneum and recombinant chromosomes showed the most marked tendency to produce laggards in both divisions. Future attempts to advance knowledge on sugarcane genetics and genomics should take meiotic chromosome behaviour information into account.     

Molecular investigation of the genetic base of sugarcane cultivars

 Molecular diversity was analysed among 162 clones of sugarcane using DNA restriction fragment length polymorphism (RFLP). One hundred and nine of them were modern cultivars of interspecific origin; most of them were bred in Barbados or in Mauritius. Fifty three were from Saccharum officinarum species, which is the major source of genes in modern cultivars, prevailing over the part of the genome incorporated from the wild species Saccharum spontaneum. Twelve low-copy nuclear DNA probes scattered over the genome were used in combination with one or two restriction enzymes. A total of 399 fragments was identified, 386 of which were polymorphic. Each sugarcane clone displayed a high number of fragments per probe/enzyme combination, illustrating the polyploid constitution of the genome. Among the S. officinarum clones, those from New Guinea had the largest variability and encompassed that present among clones collected from the Indonesian Islands and those known to have been involved in the parentage of modern cultivars. This is in agreement with the hypothesis that New Guinea is the centre of origin of this species. The clones from New Caledonia formed a separate group and could correspond to S. officinarum clones modified through introgression with other members of the ‘Saccharum complex’. Despite the low number of S. officinarum clones used for breeding cultivars, more than 80% of the markers present in the whole S. officinarum sample were also found in modern cultivars due probably to a high heterozygosity related to polyploidy. Among the cultivars, the two main groups, originating from Barbados and Mauritius, were clearly separated. This appeared essentially due to S. spontaneum alleles present in Mauritian cultivars and absent in Barbadan ones, probably in relation to the regular use of early generation interspecific hybrids in the breeding program employed in Mauritius. Received: 9 November 1998 / Accepted: 19 November 1998     

Cytogenetics of modern sugar canes

Modern sugar cane varieties are derived from interspecific crosses involving as many as four species. Because a chromosome increase accompanies certain crosses and backcrosses, modern varieties have very high aneuploid chromosome numbers and complicated genetics. Despite this complexity, the chromosome behavior of some modern varieties approaches that of allopolyploids. In achieving homozygosity, therefore, such varieties should respond to inbreeding almost like diploids. The meiotic chromosome behavior of F₁ hybrids and modern varieties indicates little or no genetic exchange between chromosomes ofSaccharum officinarum andS. spontaneum. Irradiation may break linkages between desirable and undesirableS. spontaneum genes not ordinarily broken by crossing-over between the chromosomes of the two species. The quick success of nobilizingS. spontaneum (recurrently back-crossing to “noble canes”) depends on a peculiar increase of the chromosomes ofS. officinarum. Experience with nobilizingS. spontaneum should not make breeders impatient when they turn to interspecific crosses unaccompanied by chromosome increases.     

Biochemical genetic markers in sugarcane

Summary Isozyme variation was used to identify biochemical markers of potential utility in sugarcane genetics and breeding. Electrophoretic polymorphism was surveyed for nine enzymes among 39 wild and noble sugarcane clones, belonging to the species most closely related to modern varieties. Up to 114 distinct bands showing presence versus absence type of variation were revealed and used for qualitative characterization of the materials. Multivariate analysis of the data isolated the Erianthus clone sampled and separated the Saccharum spontaneum clones from the S. robustum and S. officinarum clones; the latter two were not differentiated from one another. The analysis of self-progenies of a 2n=112 S. spontaneum and of a commercial variety showed examples of mono- and polyfactorial segregations. Within the progeny of the variety, co-segregation of two isozymes frequent in S. spontaneum led to them being assigned to a single chromosome initially contributed by a S. spontaneum donor. This illustrates how combined survey of ancestral species and segregation analysis in modern breeding materials should permit using the lack of interspecific cross-over to establish linkage groups in a sugarcane genome.     

Triterpene methyl ethers in leaf waxes of Saccharum and related genera

The triterpene methyl ethers in the leaf waxes of over 80 clones of Saccharum officinarum, S. edule, S. robustum, S. spontaneum and a limited number of related species were compared as possible chemotaxonomic markers by GLC. The principal components were arundoin, crusgallin and cylindrin. The overall interspecific variation was small, but arundoin was particularly characteristic of S. officinarum. However, each species showed marked interclonal variation, which was related to chromosome numbers and geographical origin. Most S. spontaneum clones from India were atypical containing no triterpene methyl ethers.     

Sugarcane genome architecture decrypted with chromosome‐specific oligo probes

Sugarcane (Saccharum spp.) is probably the crop with the most complex genome. Modern cultivars (2n = 100–120) are highly polyploids and aneuploids derived from interspecific hybridization between Saccharum officinarum (2n = 80) and Saccharum spontaneum (2n = 40–128). Chromosome‐specific oligonucleotide probes were used in combination with genomic in situ hybridization to analyze the genome architecture of modern cultivars and representatives of their parental species. The results validated a basic chromosome number of x = 10 for S. officinarum. In S. spontaneum, rearrangements occurred from a basic chromosome of x = 10, probably in the Northern part of India, in two steps leading to x = 9 and then x = 8. Each step involved three chromosomes that were rearranged into two. Further polyploidization led to the wide geographical extension of clones with x = 8. We showed that the S. spontaneum contribution to modern cultivars originated from cytotypes with x = 8 and varied in proportion between cultivars (13–20%). Modern cultivars had mainly 12 copies for each of the first four basic chromosomes, and a more variable number for those basic chromosomes whose structure differs between the two parental species. One?four of these copies corresponded to entire S. spontaneum chromosomes or interspecific recombinant chromosomes. In addition, a few inter‐chromosome translocations were revealed. The new information and cytogenetic tools described in this study substantially improve our understanding of the extreme level of complexity of modern sugarcane cultivar genomes.     

Molecular cytogenetic investigation of chromosome composition and transmission in sugarcane

Modern sugarcane cultivars (Saccharum spp., 2n = 100–120) are complex polyploids derived from interspecific hybridization performed a century ago between the sugar-producing species S. officinarum L. and the wild species S. spontaneum L. Using genomic in situ hybridization, we revealed that between 15 and 27.5% of the genome of modern cultivars is derived from S. spontaneum, including 10–23% of entire chromosomes from this wild species and 8–13% chromosomes derived from interspecific recombination. We confirmed the occurrence of 2n + n transmission in crosses and first backcrosses between these two species and demonstrated that this also can occur in crosses between S. officinarum and modern cultivars. We analysed five S. officinarum clones with more than 80 chromosomes and demonstrated that they were derived from interspecific hybridization supporting the classical view that this species is characterized by 2n = 80. We also illustrated the complementarities between molecular cytogenetics and genetic mapping approaches for analysing complex genomes.     

Genetic dissection of a modern sugarcane cultivar (Saccharum spp.). I. Genome mapping with AFLP markers

Sugarcane cultivars are polyploid, aneuploid clones derived from interspecific hybridization between Saccharum officinarum and S. spontaneum. Their genome has recently started to be unravelled as a result of the development of molecular markers. We constructed an AFLP genetic map based on a selfing population of a specific cultivar, R570.Using 37 AFLP primer pairs, we detected 1,185 polymorphic markers of which 939 were simplex (segregated 3:1); these were used to construct the map. Of those 939, 887 were distributed on 120 cosegregation groups (CGs) based on linkages in coupling, while 52 remained unlinked. The cumulative length of all the groups was 5,849 cM, which is probably around one-third of the total genome length. Comparison with reference S. officinarum clones enabled us to assign 11 and 79 CGs to S. spontaneum and S. officinarum,respectively, whereas 11 CGs were probably derived from recombination between chromosomes of the two ancestral species. The patchy size of the groups, which ranges from 1 to 232 cM, illustrates the difficulty to access large portions of chromosomes, particularly those inherited from S. officinarum. Repulsion phase linkages suggested a high preferential pairing for 13 CG pairs. Out of the 120 CGs, 34 could be assigned to one of the 10 homo(eo)logy groups already defined in a previous RFLP map owing to the use of a small common marker set. The genome coverage was significantly increased in the map reported here. Implications for quantitative trait loci (QTL) research and marker-assisted breeding perspectives are discussed. Received: 31 August 2000 / Accepted: 16 October 2000     

Characterisation of the double genome structure of modern sugarcane cultivars (Saccharum spp.) by molecular cytogenetics

Cultivated sugarcane clones (Saccharum spp., 2n=100 to 130) are derived from complex interspecific hybridizations between the speciesS. officinarum andS. spontaneum. Using comparative genomic DNA in situ hybridization, we demonstrated that it is possible to distinguish the chromosomes contributed by these two species in an interspecific F1 hybrid and a cultivated clone, R570. In the interspecific F1 studied, we observed n+n transmission of the parental chromosomes instead of the peculiar 2n+n transmission usually described in such crosses. Among the chromosomes of cultivar R570 (2n=107–115) about 10% were identified as originating fromS. spontaneum and about 10% were identified as recombinant chromosomes between the two speciesS. officinarum andS. spontaneum. This demonstrated for the first time the occurrence of recombination between the chromosomes of these two species. The rDNA sites were located by in situ hybridization in these two species and the cultivar R570. This supported different basic chromosome numbers and chromosome structural differences between the two species and provided a first bridge between physical and genetical mapping in sugarcane.     

Complete chloroplast genomes of Saccharum spontaneum,Saccharum officinarum and Miscanthus floridulus (Panicoideae: Andropogoneae) reveal the plastid view on sugarcane origins

Sugarcane (Saccharum hybrid cultivar) ranks among the world's top 10 food crops and annually provides 60–70% of the sugar produced worldwide. Despite its economic importance there has been no large-scale systematics study of genus Saccharum and the existing model of sugarcane origins has remained largely unchallenged for almost 50 years. For the first time, we have assembled the complete plastid genomes of Miscanthus floridulus (first report for this genus), Saccharum spontaneum and Saccharum officinarum allowing us to elucidate the phylogenetic origins of Saccharum s.s. species. We demonstrate that Saccharum s.s. is divided into four species, with S. spontaneum diverging from the remainder of the genus about 1.5 million years ago and S. robustum diverging 750,000 years ago. Two separate lineages, one leading to S. officinarum and the other leading to modern hybrid cultivars diverged from S. robustum 640,000 years ago. These findings overturn all previous hypotheses on sugarcane origins, demonstrating that sugarcane's antecedents could not have arisen by human action. All modern cultivars share a common Polynesian origin, whereas Old World canes, S. barberi and S. sinense, cluster as a distinct S. officinarum lineage. This makes modern cultivars a distinct species of genus Saccharum, and we formally propose the name Saccharum cultum for the ancestor of all lineages currently classified as Saccharum hybrid cultivars.     

Characterisation of the double genome structure of modern sugarcane cultivars (Saccharum spp.) by molecular cytogenetics

Cultivated sugarcane clones (Saccharum spp., 2n=100 to 130) are derived from complex interspecific hybridizations between the speciesS. officinarum andS. spontaneum. Using comparative genomic DNA in situ hybridization, we demonstrated that it is possible to distinguish the chromosomes contributed by these two species in an interspecific F1 hybrid and a cultivated clone, R570. In the interspecific F1 studied, we observed n+n transmission of the parental chromosomes instead of the peculiar 2n+n transmission usually described in such crosses. Among the chromosomes of cultivar R570 (2n=107–115) about 10% were identified as originating fromS. spontaneum and about 10% were identified as recombinant chromosomes between the two speciesS. officinarum andS. spontaneum. This demonstrated for the first time the occurrence of recombination between the chromosomes of these two species. The rDNA sites were located by in situ hybridization in these two species and the cultivar R570. This supported different basic chromosome numbers and chromosome structural differences between the two species and provided a first bridge between physical and genetical mapping in sugarcane.     

Sugarcane Underground Organs: Going Deep for Sustainable Production

Sugarcane breeding has greatly advanced in recent decades, but many aspects of sugarcane physiology are still poorly understood, including the root-shoot relationships that ultimately affect yield. Traditional methods for studying root systems are imprecise due to methodological difficulties of in situ assessment and sampling; this seems especially true for the sugarcane root system. Studies on sugarcane roots lag well behind those on other crops, in part due to the large plant stature and long crop cycle. Commercial sugarcane cultivars are hybrids from crosses mostly between Saccharum officinarum and S. spontaneum made by breeders at the beginning of the last century. These hybrids have a genomic structure composed of 80% S. officinarum, 10% S. spontaneum and 10% recombinants of these two species. S. spontaneum is included in large part for the robustness of its underground organs (root and rhizome). The S. spontaneum genes controlling these characteristics may be lost during recurrent backcrosses with S. officinarum to increase sugar content and yield. Thus, ratooning ability is one of the most desired traits. Ratooning ability comes mainly from the rhizomatousness of S. spontaneum, but this trait has been diluted during the selection process so that the stubble of hybrids does not have rhizomes sensu stricto. In this review, we revisit some basic aspects of the sugarcane root system, mainly from an ecophysiological view, and point out considerations for breeders to consider in designing the architecture of a new sugarcane cultivar that can meet the need for sustainable agricultural production.     

The Sugarcane Genome Challenge: Strategies for Sequencing a Highly Complex Genome

Sugarcane cultivars derive from interspecific hybrids obtained by crossing Saccharum officinarum and Saccharum spontaneum and provide feedstock used worldwide for sugar and biofuel production. The importance of sugarcane as a bioenergy feedstock has increased interest in the generation of new cultivars optimised for energy production. Cultivar improvement has relied largely on traditional breeding methods, which may be limited by the complexity of inheritance in interspecific polyploid hybrids, and the time-consuming process of selection of plants with desired agronomic traits. In this sense, molecular genetics can assist in the process of developing improved cultivars by generating molecular markers that can be used in the breeding process or by introducing new genes into the sugarcane genome. For meeting each of these, and additional goals, biotechnologists would benefit from a reference genome sequence of a sugarcane cultivar. The sugarcane genome poses challenges that have not been addressed in any prior sequencing project, due to its highly polyploid and aneuploid genome structure with a complete set of homeologous genes predicted to range from 10 to 12 copies (alleles) and to include representatives from each of two different species. Although sugarcane’s monoploid genome is about 1 Gb, its highly polymorphic nature represents another significant challenge for obtaining a genuine assembled monoploid genome. With a rich resource of expressed-sequence tag (EST) data in the public domain, the present article describes tools and strategies that may aid in the generation of a reference genome sequence.     

Three founding ancestral genomes involved in the origin of sugarcane

Background and AimsModern sugarcane cultivars (Saccharum spp.) are high polyploids, aneuploids (2n = ~12x = ~120) derived from interspecific hybridizations between the domesticated sweet species Saccharum officinarum and the wild species S. spontaneum.MethodsTo analyse the architecture and origin of such a complex genome, we analysed the sequences of all 12 hom(oe)ologous haplotypes (BAC clones) from two distinct genomic regions of a typical modern cultivar, as well as the corresponding sequence in Miscanthus sinense and Sorghum bicolor, and monitored their distribution among representatives of the Saccharum genus.Key ResultsThe diversity observed among haplotypes suggested the existence of three founding genomes (A, B, C) in modern cultivars, which diverged between 0.8 and 1.3 Mya. Two genomes (A, B) were contributed by S. officinarum; these were also found in its wild presumed ancestor S. robustum, and one genome (C) was contributed by S. spontaneum. These results suggest that S. officinarum and S. robustum are derived from interspecific hybridization between two unknown ancestors (A and B genomes). The A genome contributed most haplotypes (nine or ten) while the B and C genomes contributed one or two haplotypes in the regions analysed of this typical modern cultivar. Interspecific hybridizations likely involved accessions or gametes with distinct ploidy levels and/or were followed by a series of backcrosses with the A genome. The three founding genomes were found in all S. barberi, S. sinense and modern cultivars analysed. None of the analysed accessions contained only the A genome or the B genome, suggesting that representatives of these founding genomes remain to be discovered.ConclusionsThis evolutionary model, which combines interspecificity and high polyploidy, can explain the variable chromosome pairing affinity observed in Saccharum. It represents a major revision of the understanding of Saccharum diversity.     

Differential detection of transposable elements between Saccharum species

Cultivars of sugarcane (Saccharum) are hybrids between species S. officinarum (x = 10, 2n = 8x = 80) and S. spontaneum (x = 8, 2n = 5 – 16x = 40 – 128). These accessions have 100 to 130 chromosomes, 80–85% of which are derived from S. officinarum, 10–15% from S. spontaneum, and 5–10% are possible recombinants between the two genomes. The aim of this study was to analyze the repetition of DNA sequences in S. officinarum and S. spontaneum. For this purpose, genomic DNA from S. officinarum was digested with restriction enzymes and the fragments cloned. Sixty-eight fragments, approximately 500 bp, were cloned, sequenced and had their identity analyzed in NCBI, and in the rice, maize, and sorghum genome databases using BLAST. Twelve clones containing partial transposable elements, one single-copy control, one DNA repetitive clone control and two genome controls were analyzed by DNA hybridization on membrane, using genomic probes from S. officinarum and S. spontaneum. The hybridization experiment revealed that six TEs had a similar repetitive DNA pattern in the genomes of S. officinarum and S. spontaneum, while six TEs were more abundant in the genome of S. officinarum. We concluded that the species S. officinarum and S. spontaneum have differential accumulation LTR retrotransposon families, suggesting distinct insertion or modification patterns.     

A comprehensive genetic map of sugarcane that provides enhanced map coverage and integrates high-throughput Diversity Array Technology (DArT) markers

Background

Sugarcane genetic mapping has lagged behind other crops due to its complex autopolyploid genome structure. Modern sugarcane cultivars have from 110-120 chromosomes and are in general interspecific hybrids between two species with different basic chromosome numbers: Saccharum officinarum (2n = 80) with a basic chromosome number of 10 and S. spontaneum (2n = 40-128) with a basic chromosome number of 8. The first maps that were constructed utilised the single dose (SD) markers generated using RFLP, more recent maps generated using AFLP and SSRs provided at most 60% genome coverage. Diversity Array Technology (DArT) markers are high throughput allowing greater numbers of markers to be generated.

Results

Progeny from a cross between a sugarcane variety Q165 and a S. officinarum accession IJ76-514 were used to generate 2467 SD markers. A genetic map of Q165 was generated containing 2267 markers, These markers formed 160 linkage groups (LGs) of which 147 could be placed using allelic information into the eight basic homology groups (HGs) of sugarcane. The HGs contained from 13 to 23 LGs and from 204 to 475 markers with a total map length of 9774.4 cM and an average density of one marker every 4.3 cM. Each homology group contained on average 280 markers of which 43% were DArT markers 31% AFLP, 16% SSRs and 6% SNP markers. The multi-allelic SSR and SNP markers were used to place the LGs into HGs.

Conclusions

The DArT array has allowed us to generate and map a larger number of markers than ever before and consequently to map a larger portion of the sugarcane genome. This larger number of markers has enabled 92% of the LGs to be placed into the 8 HGs that represent the basic chromosome number of the ancestral species, S. spontaneum. There were two HGs (HG2 and 8) that contained larger numbers of LGs verifying the alignment of two sets of S. officinarum chromosomes with one set of S. spontaneum chromosomes and explaining the difference in basic chromosome number between the two ancestral species. There was also evidence of more complex structural differences between the two ancestral species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-152) contains supplementary material, which is available to authorized users.     

Chromosome assortment in Saccharum

Recent work has revealed random chromosome pairing and assortment in Saccharum spontaneum L., the most widely distributed, and morphologically and cytologically variable of the species of Saccharum. This conclusion was based on the analysis of a segregating population from across between S. spontaneum SES 208 and a spontaneously-doubled haploid of itself, derived from anther culture. To determine whether polysomic inheritance is common in Saccharum and whether it is observed in a typical biparental cross, we studied chromosome pairing and assortment in 44 progeny of a cross between euploid, meiotically regular, 2n=80 forms of Saccharum officinarum LA Purple and Saccharum robustum Mol 5829. Papuan 2n=80 forms of S. robustum have been suggested as the immediate progenitor species for cultivated sugarcane (S. officinarum). A total of 738 loci in LA Purple and 720 loci in Mol 5829 were amplified and typed in the progeny by arbitrarily primed PCR using 45 primers. Fifty and 33 single-dose polymorphisms were identified in the S. officinarum and S. robustum genomes, respectively ( ₂ at 98%). Linkage analysis of single-dose polymorphisms in both genomes revealed linkages in repulsion and coupling phases. In the S. officinarum genome, a map hypothesis gave 7 linkage groups with 17 linked and 33 unlinked markers. Four of 13 pairwise linkages were in repulsion phase and 9 were in coupling phase. In the S. robustum genome, a map hypothesis gave 5 linkage groups, defined by 12 markers, with 21 markers unlinked, and 2 of 9 pairwise linkages were in repulsion phase. Therefore, complete polysomic inheritance was not observed in either species, suggesting that chromosomal behavior is different from that observed by linkage analysis of over 500 markers in the S. spontaneum map. Implications of this finding for evolution and breeding are discussed.     

A contribution to the cyto-taxonomy of Brassica (Cruciferae) and its allies

Material of 85 species of Brassica and genera closely related to it was examined cytologically, and the chroiftosome counts are reported. These confirm records published by other workers for 42 species, two contradict earlier records, and 41 counts are believed to be new. Different species with the same chromosome number have been cross-pollinated, and those found to be interfertile have been grouped into cytodemes. For the most part species found to be interfertile have long been regarded as closely akin, but three cases of interfertile intergeneric hybrids are reported. Altogether the material has been classified into 45 cytodemes: 35 diploids with chromosome numbers ranging from n= 7 to n= 13, and 10 derived tetraploids. This enumeration is possibly not exhaustive of the world's genomes closely related to the crop Brassicas and a list is appended of the wild species which need examination before the enumeration might be considered to be complete.