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.

ESTs as a source for sequence polymorphism discovery in sugarcane: example of the Adh genes.

Expressed sequence tags (ESTs) have proven to be a valuable tool to discover single nucleotide polymorphism (SNP) in human genes but their use for this purpose is still limited in higher plants. Using a database of approximately 250,000 sugarcane ESTs we have recovered 219 sequences encoding alcohol dehydrogenases ( Adh), which tagged 178 distinct cDNAs from 27 libraries, constructed from at least four different cultivars. The partitioning of these ESTs into paralogous genes revealed three Adh genes expressed in sugarcane, one Adh2 and two Adh1. The soundness of the partition was carefully checked by comparison to external data, especially from the closely related sorghum. Analysis of polymorphism in the alignments of EST sequences revealed a total of 37 highly reliable SNPs in the coding and untranslated regions of the three Adh genes. In the coding regions, the mean occurrence of SNPs was one for every 122 base pair. A total of eight insertion-deletions was observed, their occurrence being limited to untranslated regions. These results show that EST data constitute an invaluable source of sequence polymorphism for sugarcane that is worth carefully collecting for the future development of new marker tools.

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.

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.

A putative major gene for rust resistance linked with a RFLP marker in sugarcane cultivar 'R570'.

Inheritance of resistance to rust was investigated in the self progeny of the sugarcane cultivar 'R570' also used to build a RFLP genetic map. Resistance was evaluated through both field and controlled greenhouse trials. A clear-cut 3 (resistant) ∶ 1 (susceptible) segregation indicative of a probable dominant resistant gene was observed. This is the first documented report of a monogenic inheritance for disease resistance in sugarcane. This gene was found linked at 10 cM with an RFLP marker revealed by probe CDSR29. Other minor factors involved in the resistance were also detected.

Identification and characterisation of sugarcane intergeneric hybrids, Saccharum officinarum x Erianthus arundinaceus, with molecular markers and DNA in situ hybridisation.

Molecular markers were used to characterise sugarcane intergeneric hybrids between S. officinarum and E. arundinaceus. Very simple diagnostic tools for hybrid identification among the progeny were derived from isozyme electrophoresis and a sequence-tagged PCR. Two enzyme systems (GOT and MDH B) and PCR amplification revealing spacer-size variation in the 5s-rDNA cluster were found most convenient. Specific characterisation of the two genomic components was possible using RFLP and in situ hybridisation. The strong molecular differentiation between S. officinarum and E. arundinaceus allows the identification of numerous Erianthus-specific RFLP bands in the hybrids. Genomic DNA in situ hybridisation allows for the differentiation of the chromosomes contributed by S. officinarum and E. arundinaceus in chromosome preparations of the hybrids. In situ hybridisation with the 18s-5.8s-25s rDNA probe highlights the basic chromosome numbers in the two parental species. The potential of these techniques to monitor the Erianthus genome during the introgression process is discussed.

A molecular approach to unraveling the genetics of sugarcane, a complex polyploid of the Andropogoneae tribe.

Modern sugarcane varieties are complex aneuploids and typically have chromosome numbers in the 100-125 range with about 5-10% of them contributed by wild relatives, mainly Saccharum spontaneum, and the rest by S. officinarum. This particular genomic constitution was found favorable for mapping the S. spontaneum genome, using maize as a diploid reference for comparison. We conducted an analysis of 32 individuals derived from the selfing of variety SP 701006 using four isozymes and 53 maize probes which covered the whole maize genome. A total of 348 segregating bands were generated. Highly significant cosegregations enabled us to place 94 markers into 25 cosegregation groups. Eighteen of these groups involved S. spontaneum specific markers and might therefore mark S. spontaneum chromosomes in segregation. On the basis of probes in common, the 25 cosegregation groups could be assembled into eight tentative linkage groups, of which seven describe S. spontaneum chromosomes. A large degree of synteny between sugarcane and maize could be inferred, with a much lower rate of recombination in sugarcane.