The complex polyploid genome architecture of sugarcane.

Sugarcane, the world's most harvested crop by tonnage, has shaped global history, trade and geopolitics, and is currently responsible for 80% of sugar production worldwide1. While traditional sugarcane breeding methods have effectively generated cultivars adapted to new environments and pathogens, sugar yield improvements have recently plateaued2. The cessation of yield gains may be due to limited genetic diversity within breeding populations, long breeding cycles and the complexity of its genome, the latter preventing breeders from taking advantage of the recent explosion of whole-genome sequencing that has benefited many other crops. Thus, modern sugarcane hybrids are the last remaining major crop without a reference-quality genome. Here we take a major step towards advancing sugarcane biotechnology by generating a polyploid reference genome for R570, a typical modern cultivar derived from interspecific hybridization between the domesticated species (Saccharum officinarum) and the wild species (Saccharum spontaneum). In contrast to the existing single haplotype ('monoploid') representation of R570, our 8.7 billion base assembly contains a complete representation of unique DNA sequences across the approximately 12 chromosome copies in this polyploid genome. Using this highly contiguous genome assembly, we filled a previously unsized gap within an R570 physical genetic map to describe the likely causal genes underlying the single-copy Bru1 brown rust resistance locus. This polyploid genome assembly with fine-grain descriptions of genome architecture and molecular targets for biotechnology will help accelerate molecular and transgenic breeding and adaptation of sugarcane to future environmental conditions.

Prospecting sugarcane resistance to Sugarcane yellow leaf virus by genome-wide association.

KEY MESSAGE: Using GWAS approaches, we detected independent resistant markers in sugarcane towards a vectored virus disease. Based on comparative genomics, several candidate genes potentially involved in virus/aphid/plant interactions were pinpointed. Yellow leaf of sugarcane is an emerging viral disease whose causal agent is a Polerovirus, the Sugarcane yellow leaf virus (SCYLV) transmitted by aphids. To identify quantitative trait loci controlling resistance to yellow leaf which are of direct relevance for breeding, we undertook a genome-wide association study (GWAS) on a sugarcane cultivar panel (n = 189) representative of current breeding germplasm. This panel was fingerprinted with 3,949 polymorphic markers (DArT and AFLP). The panel was phenotyped for SCYLV infection in leaves and stalks in two trials for two crop cycles, under natural disease pressure prevalent in Guadeloupe. Mixed linear models including co-factors representing population structure fixed effects and pairwise kinship random effects provided an efficient control of the risk of inflated type-I error at a genome-wide level. Six independent markers were significantly detected in association with SCYLV resistance phenotype. These markers explained individually between 9 and 14 % of the disease variation of the cultivar panel. Their frequency in the panel was relatively low (8-20 %). Among them, two markers were detected repeatedly across the GWAS exercises based on the different disease resistance parameters. These two markers could be blasted on Sorghum bicolor genome and candidate genes potentially involved in plant-aphid or plant-virus interactions were localized in the vicinity of sorghum homologs of sugarcane markers. Our results illustrate the potential of GWAS approaches to prospect among sugarcane germplasm for accessions likely bearing resistance alleles of significant effect useful in breeding programs.

Mutator System Derivatives Isolated from Sugarcane Genome Sequence.

Mutator-like transposase is the most represented transposon transcript in the sugarcane transcriptome. Phylogenetic reconstructions derived from sequenced transcripts provided evidence that at least four distinct classes exist (I-IV) and that diversification among these classes occurred early in Angiosperms, prior to the divergence of Monocots/Eudicots. The four previously described classes served as probes to select and further sequence six BAC clones from a genomic library of cultivar R570. A total of 579,352 sugarcane base pairs were produced from these "Mutator system" BAC containing regions for further characterization. The analyzed genomic regions confirmed that the predicted structure and organization of the Mutator system in sugarcane is composed of two true transposon lineages, each containing a specific terminal inverted repeat and two transposase lineages considered to be domesticated. Each Mutator transposase class displayed a particular molecular structure supporting lineage specific evolution. MUSTANG, previously described domesticated genes, are located in syntenic regions across Sacharineae and, as expected for a host functional gene, posses the same gene structure as in other Poaceae. Two sequenced BACs correspond to hom(eo)logous locus with specific retrotransposon insertions that discriminate sugarcane haplotypes. The comparative studies presented, add information to the Mutator systems previously identified in the maize and rice genomes by describing lineage specific molecular structure and genomic distribution pattern in the sugarcane genome. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12042-012-9104-y) contains supplementary material, which is available to authorized users.

Haplotype structure around Bru1 reveals a narrow genetic basis for brown rust resistance in modern sugarcane cultivars.

Modern sugarcane cultivars (Saccharum spp., 2n = 100-130) are high polyploid, aneuploid and of interspecific origin. A major gene (Bru1) conferring resistance to brown rust, caused by the fungus Puccinia melanocephala, has been identified in cultivar R570. We analyzed 380 modern cultivars and breeding materials covering the worldwide diversity with 22 molecular markers genetically linked to Bru1 in R570 within a 8.2 cM segment. Our results revealed a strong LD in the Bru1 region and strong associations between most of the markers and rust resistance. Two PCR markers, that flank the Bru1-bearing segment, were found completely associated with one another and only in resistant clones representing efficient molecular diagnostic for Bru1. On this basis, Bru1 was inferred in 86 % of the 194 resistant sugarcane accessions, revealing that it constitutes the main source of brown rust resistance in modern cultivars. Bru1 PCR diagnostic markers should be particularly useful to identify cultivars with potentially alternative sources of resistance to diversify the basis of brown rust resistance in breeding programs.

Genetics of sex determination in tilapiine species.

We identified DNA markers linked to sex determining genes in six closely related species of tilapiine fishes. The mode of sex determination differed among species. In Oreochromis karongae and Tilapia mariae the sex-determining locus is on linkage group (LG) 3 and the female is heterogametic (WZ-ZZ system). In O. niloticus and T. zillii the sex-determining locus is on LG1 and the male is heterogametic (XX-XY system). A more complex pattern was observed in O. aureus and O. mossambicus, in which markers on both LG1 and LG3 were associated with sex. We found evidence for sex-linked lethal effects on LG1, as well as interactions between loci in the two linkage groups. Comparison of genetic and physical maps demonstrated a broad region of recombination suppression harboring the sex-determining locus on LG3. Sex-specific recombination suppression was found in the female heterogametic sex. Sequence analysis showed the accumulation of repetitive elements in this region. Phylogenetic analysis suggests that at least two transitions in the mode of sex determination have occurred in this clade. This variation in sex determination mechanisms among closely related species makes tilapias an excellent model system for studying the evolution of sex chromosomes in vertebrates.

Genetic mapping in sugarcane, a high polyploid, using bi-parental progeny: identification of a gene controlling stalk colour and a new rust resistance gene.

Modern sugarcane cultivars (Saccharum spp) are highly polyploïd and aneuploid interspecific hybrids (2n = 100-130). Two genetic maps were constructed using a population of 198 progeny from a cross between R570, a modern cultivar, and MQ76-53, an old Australian clone derived from a cross between Trojan (a modern cultivar) and SES528 (a wild Saccharum spontaneum clone). A total of 1,666 polymorphic markers were produced using 37 AFLP primer combinations, 46 SSRs and 9 RFLP probes. Linkage analysis led to the construction of 86 cosegregation groups for R570 and 105 cosegregation groups for MQ76-53 encompassing 424 and 536 single dose markers, respectively. The cumulative length of the R570 map was 3,144 cM, while that of the MQ76-53 map was 4,329 cM. Here, we integrated mapping information obtained on R570 in this study with that derived from a previous map based on a selfed R570 population. Two new genes controlling Mendelian traits were localized on the MQ76-53 map: a gene controlling the red stalk colour was linked at 6.5 cM to an AFLP marker and a new brown rust resistance gene was linked at 23 cM to an AFLP marker. Besides another previously identified brown rust resistance gene (Bru1), these two genes are the only other major genes to be identified in sugarcane so far.

Unraveling the genome structure of polyploids using FISH and GISH; examples of sugarcane and banana.

We review here the progress that has been achieved using molecular cytogenetics to analyze the genome structure of sugarcane (Saccharum spp) and banana (Musa spp), two crops that are polyploid, of interspecific origin and with chromosomes not distinguishable by their gross morphology. In Saccharum, molecular cytogenetics enabled us to determine the basic chromosome number of two species, Saccharum officinarum and S. spontaneum, involved in the origin of modern cultivars, to quantify the proportion of chromosomes of these species in the genome of modern cultivars, to assess the extent of interspecific chromosome recombination and to clarify the origin of the related species S. barberi. These techniques are also used to monitor introgression with related genera. In Musa, GISH enabled us to differentiate the four genomes involved in banana cultivars and allowed us to determine the genome constitution of several cultivars. FISH was used to analyze the distribution of repeated sequences along the genome.

Differential chromosome pairing affinities at meiosis in polyploid sugarcane revealed by molecular markers.

Chromosome pairing at meiosis is an essential feature in cell biology, which determines trait inheritance and species evolution. Complex polyploids may display diverse pairing affinities and offer favorable situations for studying meiosis. The genus Saccharum encompasses diverse forms of polyploids with predominantly bivalent pairing. We have focused on a modern cultivar of sugarcane, R570, and taken advantage of a particular single copy probe (BNL 12.06) revealing 11 alleles by restriction fragment length polymorphism (RFLP). As for other cultivars, R570 is highly polyploid (2n=ca. 115) and indirectly derived from interspecific hybridization between Saccharum officinarum (2n=80, x=10) and S. spontaneum (2n=40-128, x=8). Here we determined the doses of the various BNL12.06 RFLP alleles among 282 progeny of R570 and estimated the mutual pairing frequencies among the corresponding homo- or homoeologous chromosomes using a maximum likelihood method. The result is an atypical picture, with pairing frequencies ranging from 0 to 40% and differential affinities leading to the identification of several chromosome subsets. This example illustrates the unsystematic meiotic behavior in a complex polyploid. It highlights a continuous range of pairing affinities between chromosomes and pinpoints a strong role of individual chromosome features, partly related to their ancestral origin, in the determination of these affinities.

Targeted mapping of a sugarcane rust resistance gene (Bru1) using bulked segregant analysis and AFLP markers.

The presence of a major resistance gene (Bru1) for brown rust in the sugarcane cultivar R570 (2n about 115) was confirmed by analyzing segregation of rust resistance in a large population of 658 individuals, derived from selfing of clone R570. A subset of this population was analyzed with AFLP and bulked segregant analysis (BSA) to develop a detailed genetic map around the resistance gene. Four hundred and forty three primer pairs were used resulting in the identification of eight AFLP markers surrounding the resistance gene in an interval of 10 cM, with the closest markers located at 1.9 and 2.2 cM on each side of the gene. Efficiency of the AFLP/BSA applied to the complex polyploid genome of sugarcane is discussed, as well as the potential of the newly identified AFLP markers for developing a map-based cloning approach exploiting, synteny conservation with sorghum.

Genomic distribution and characterization of EST-derived resistance gene analogs (RGAs) in sugarcane.

A large sugarcane EST (expressed sequence tag) project recently gave us access to 261,609 EST sequences from sugarcane, assembled into 81,223 clusters. Among these, we identified 88 resistance gene analogs (RGAs) based on their homology to typical pathogen resistance genes, using a stringent BLAST search with a threshold e-value of e(-50). They included representatives of the three major groups of resistance genes with NBS/LRR, LRR or S/T KINASE domains. Fifty RGAs showed a total of 148 single-dose polymorphic RFLP markers, which could be located on the sugarcane reference genetic map (constructed in cultivar R570, 2n=approximately 115). Fifty-five SSR loci corresponding to 134 markers in R570 were also mapped to enable the classification of the various haplotypes into homology groups. Several RGA clusters were found. One cluster of two LRR-like loci mapped close to the only disease resistance gene known so far in sugarcane, which confers resistance to common rust. Detailed sequence comparison between two NBS/LRR RGA clusters in relation to their orthologs in rice and maize suggests their polyphyletic origins, and indicates that the degree of divergence between paralogous RGAs in sugarcane can be larger than that from an ortholog in a distant species.

Characterisation of the phosphoenolpyruvate carboxylase gene family in sugarcane (Saccharum spp.).

Phosphoenolpyruvate carboxylases (PEPCs) are encoded by a small multigenic family. In order to characterise this gene family in sugarcane, seven DNA fragments displaying a high homology with grass PEPC genes were isolated using polymerase chain reaction-based cloning. A phylogenetic study revealed the existence of four main PEPC gene lineages in grasses and particularly in sugarcane. Moreover, this analysis suggests that grass C4 PEPC has likely derived from a root pre-existing isoform in an ancestral species. Using the Northern-dot-blot method, we studied the expression of the four PEPC gene classes in sugarcane cv. R570. We confirmed that transcript accumulation of the C4 PEPC gene (ppc-C4) mainly occurs in the green leaves and is light-induced. We also showed that another member of this gene family (ppc-aR) is more highly transcribed in the roots. The constitutive expression for a previously characterised gene (ppc-aL2) was confirmed. Lastly, the transcript accumulation of the fourth PEPC gene class (ppc-aL1) was not revealed. Length polymorphism in non-coding regions for three PEPC gene lineages enabled us to develop sequence-tagged site PEPC markers in sugarcane. We analysed the segregation of PEPC fragments in self-pollinated progenies of cv. R570 and found co-segregating fragments for two PEPC gene lineages. This supports the hypothesis that diversification of the PEPC genes involved duplications, probably in tandem.

Construction and characterization of a bacterial artificial chromosome library of banana (Musa acuminata Colla).

A bacterial artificial chromosome (BAC) library for banana was constructed from leaves of the wild diploid 'Calcutta 4' clone (Musa acuminata subsp. Burmannicoides 2n = 2 x = 22). 'Calcutta 4' is widely used in breeding programs for its resistance to the current major disease of banana and is being used to build a genetic reference map of banana. As banana leaves are particularly rich in polyphenols and polysaccharides a protocol was adapted to isolate intact nuclei and high-molecular-weight (HMW) DNA. A total of 55,152 clones with an average insert size of 100 kb were picked. The frequency of BAC clones carrying inserts derived from chloroplast and mitochondrial DNA was estimated to be 1.5%. The coverage of the library is equivalent to 9.0-times the haploid genome. The BAC library was screened with 13 RFLP probes belonging to the 8 linkage groups of the consensus molecular map of banana. A total of 135 clones were identified giving an average of 10.38 clones for each locus. This BAC library will be a valuable starting tool for many of the goals of the recently emerged International Musa Genomic Consortium. One of our initial objectives will be to develop a banana physical map by BAC-FISH (fluorescent in situ hybridization) viewing the characterization of translocation break points.

Genetic dissection of a modern sugarcane cultivar ( Saccharum spp.).II. Detection of QTLs for yield components.

The genetics of current sugarcane cultivars ( Saccharum spp.) is outstandingly complex, due to a high ploidy level and an interspecific origin which leads to the presence of numerous chromosomes belonging to two ancestral genomes. In order to analyse the inheritance of quantitative traits, we have undertaken an extensive Quantitative Trait Allele (QTA) mapping study based on a population of 295 progenies derived from the selfing of cultivar R570, using about 1,000 AFLP markers scattered on about half of the genome. The population was evaluated in a replicated trial for four basic yield components, plant height, stalk number, stalk diameter and brix, in two successive crop-cycles. Forty putative QTAs were found for the four traits at P = 5 x 10(-3), of which five appeared in both years. Their individual size ranged between 3 and 7% of the whole variation. The stability across years was improved when limiting threshold stringency. All these results depict the presence in the genome of numerous QTAs, with little effects, fluctuating slightly across cycles, on the verge to being perceptible given the experimental resolution. Epistatic interactions were also explored and 41 independent di-genic interactions were found at P = (5 x 10(-3))(2). Altogether the putative genetic factors revealed here explain from 30 to 55% of the total phenotypic variance depending on the trait. The tentative assignment of some QTAs to the ancestral genomes showed a small majority of contributions as expected from the ancestral phenotypes. This is the first extensive QTL mapping study performed in cultivated sugarcane.

Resistance of Sugarcane Cultivar R 570 to Puccinia melanocephala Isolatesfrom Different Geographic Locations.

Two different inoculation techniques were investigated before studying the reaction of the major rust resistance gene of sugarcane cultivar R 570 against isolates of Puccinia melanocephala from different geographic locations. Cultivar R 570 exhibited severe rust symptoms when in vitro plantlets were inoculated with a rust isolate from Réunion Island, but a good correlation with field resistance was observed when detached leaves were inoculated with the pathogen. This latter technique was then used to inoculate R 570 and a sample of its self progeny with rust isolates from Brazil, Colombia, Florida (three isolates), Guadeloupe, Réunion Island, and Zimbabwe. R 570 was resistant to all isolates of P. melanocephala, and the segregation of resistance in the progeny did not change with the isolates, suggesting that a single gene, or a single chromosomic region, was involved in the resistance against all tested isolates. This major resistance gene has, therefore, potential value to improve resistance to rust in various geographic regions.

Molecular contribution to selection of intergeneric hybrids between sugarcane and the wild species Erianthus arundinaceus.

Erianthus arundinaceus has great potential as a germplasm source for better ratoonability, vigour, tolerance to environmental stresses, and disease resistance in sugarcane. Many unsuccessful attempts have been made to introduce these characters into modern sugarcane cultivars. We report on significant progress made since molecular tools were implemented. Sequence-tagged PCR, revealing size variation in the 5S rDNA cluster, was performed on intact leaf tissue to identify genuine hybrids six weeks after germination. This early screening of seedlings avoids the loss of genuine hybrids due to competition with selfed progeny. Of 96 crosses made involving female Saccharum officinarum or sugarcane cultivars (Saccharum spp.) and male E. arundinaceus, 26 were fertile producing 1328 seedlings. Thirty-seven genuine hybrids were unequivocally identified but only 19 have survived. Genuine hybrids were produced from only three crosses, all involving S. officinarum as the female parent. Chromosome elimination was observed in all seven hybrids analyzed using genomic in situ hybridization (GISH). Very little cross-hybridization was observed between the genomes of the two species after GISH, confirming recent molecular studies which showed that E. arundinaceus is quite distant from the genus Saccharum. The major limitation in the introgression of E. arundinaceus resides now in the apparent sterility of the hybrids.

Molecular characterisation and origin of the Coffea arabica L. genome.

Restriction fragment length polymorphism (RFLP) markers were used in combination with genomic in situ hybridisation (GISH) to investigate the origin of the allotetraploid species Coffea arabica (2n = 44). By comparing the RFLP patterns of potential diploid progenitor species with those of C. arabica, the sources of the two sets of chromosomes, or genomes, combined in C. arabica were identified. The genome organisation of C. arabica was confirmed by GISH using simultaneously labelled total genomic DNA from the two putative genome donor species as probes. These results clearly suggest that C. arabica is an amphidiploid formed by hybridisation between C. eugenioides and C. canephora, or ecotypes related to these diploid species. Our results also indicate low divergence between the two constituent genomes of C. arabica and those of its progenitor species, suggesting that the speciation of C. arabica took place relatively recently. Precise localisation in Central Africa of the site of the speciation of C. arabica, based on the present distribution of the coffee species, appears difficult, since the constitution and extent of tropical forest has varied considerably during the late Quaternary period.

Isolation and characterization of a satellite DNA family in the Saccharum complex.

EaCIR1, a 371-bp Erianthus-specific satellite DNA sequence, was cloned from TaqI restricted genomic DNA after agarose-gel electrophoresis. This sequence has 77% homology with a 365-bp satellite of Helictotrichon convolutum and 72% homology with a 353-bp tandem repeat sequence from Oryza sativa. PCR primers defined in the conserved regions of these repetitive sequences were used to isolate other satellite DNAs in different representatives of the Saccharum complex: SoCIR1 in Saccharum officinarum, SrCIR1 in Saccharum robustum, SsCIR1 and SsCIR2 in Saccharum spontaneum, and MsCIR1 in Miscanthus sinensis. EaCIR1 and SoCIR1 were localized to subtelomeric regions of the chromosomes by fluorescence in situ hybridization. Southern hybridization experiments, using two representatives of this repeat sequence family as probes, illustrated contrasting species-specificity and demonstrated the existence of similar repetitive elements in sorghum and maize.

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 species S. officinarum and S. 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 from S. spontaneum and about 10% were identified as recombinant chromosomes between the two species S. officinarum and S. 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.

RFLP mapping in cultivated sugarcane (Saccharum spp.): genome organization in a highly polyploid and aneuploid interspecific hybrid.

Sugarcane cultivars are polyploid, aneuploid, interspecific hybrids between the domesticated species Saccharum officinarum and the wild relative S. spontaneum. Cultivar chromosome numbers range from 100 to 130 with approximately 10% contributed by S. spontaneum. We have undertaken a mapping study on the progeny of a selfed cultivar, R570, to analyze this complex genome structure. A set of 128 restriction fragment length polymorphism probes and one isozyme was used. Four hundred and eight markers were placed onto 96 cosegregation groups, based on linkages in coupling only. These groups could tentatively be assembled into 10 basic linkage groups on the basis of common probes. Origin of markers was investigated for 61 probes and the isozyme, leading to the identification of 80 S. officinarum and 66 S. spontaneum derived markers, respectively. Their distribution in cosegregation groups showed better map coverage for the S. spontaneum than for the S. officinarum genome fraction and occasional recombination between the two genomes. The study of repulsions between markers suggested the prevalence of random pairing between chromosomes, typical of autopolyploids. However, cases of preferential pairing between S. spontaneum chromosomes were also detected. A tentative Saccharum map was constructed by pooling linkage information for each linkage group.

Comparative genetic mapping between duplicated segments on maize chromosomes 3 and 8 and homoeologous regions in sorghum and sugarcane.

Comparative mapping within maize, sorghum and sugarcane has previously revealed the existence of syntenic regions between the crops. In the present study, mapping on the sorghum genome of a set of probes previously located on the maize and sugarcane maps allow a detailed analysis of the relationship between maize chromosomes 3 and 8 and sorghum and sugarcane homoeologous regions. Of 49 loci revealed by 46 (4 sugarcane and 42 maize) polymorphic probes in sorghum, 42 were linked and were assigned to linkage groups G (28), E (10) and I (4). On the basis of common probes, a complete co-linearity is observed between sorghum linkage group G and the two sugarcane linkage groups II and III. The comparison between the consensus sorghum/sugarcane map (G/II/III) and the maps of maize chromosomes 3 and 8 reveals a series of linkage blocks within which gene orders are conserved. These blocks are interspersed with non-homoeologous regions corresponding to the central part of the two maize chromosomes and have been reshuffled, resulting in several inversions in maize compared to sorghum and sugarcane. The results emphasize the fact that duplication will considerably complicate precise comparative mapping at the whole genome scale between maize and other Poaceae.