Experimental assessment of the accuracy of genomic selection in sugarcane.

Sugarcane cultivars are interspecific hybrids with an aneuploid, highly heterozygous polyploid genome. The complexity of the sugarcane genome is the main obstacle to the use of marker-assisted selection in sugarcane breeding. Given the promising results of recent studies of plant genomic selection, we explored the feasibility of genomic selection in this complex polyploid crop. Genetic values were predicted in two independent panels, each composed of 167 accessions representing sugarcane genetic diversity worldwide. Accessions were genotyped with 1,499 DArT markers. One panel was phenotyped in Reunion Island and the other in Guadeloupe. Ten traits concerning sugar and bagasse contents, digestibility and composition of the bagasse, plant morphology, and disease resistance were used. We used four statistical predictive models: bayesian LASSO, ridge regression, reproducing kernel Hilbert space, and partial least square regression. The accuracy of the predictions was assessed through the correlation between observed and predicted genetic values by cross validation within each panel and between the two panels. We observed equivalent accuracy among the four predictive models for a given trait, and marked differences were observed among traits. Depending on the trait concerned, within-panel cross validation yielded median correlations ranging from 0.29 to 0.62 in the Reunion Island panel and from 0.11 to 0.5 in the Guadeloupe panel. Cross validation between panels yielded correlations ranging from 0.13 for smut resistance to 0.55 for brix. This level of correlations is promising for future implementations. Our results provide the first validation of genomic selection in sugarcane.

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.

Variability of grain quality in sorghum: association with polymorphism in Sh2, Bt2, SssI, Ae1, Wx and O2.

To ensure food security in Africa and Asia, developing sorghum varieties with grain quality that matches consumer demand is a major breeding objective that requires a better understanding of the genetic control of grain quality traits. The objective of this targeted association study was to assess whether the polymorphism detected in six genes involved in synthesis pathways of starch (Sh2, Bt2, SssI, Ae1, and Wx) or grain storage proteins (O2) could explain the phenotypic variability of six grain quality traits [amylose content (AM), protein content (PR), lipid content (LI), hardness (HD), endosperm texture (ET), peak gelatinization temperature (PGT)], two yield component traits [thousand grain weight (TGW) and number of grains per panicle (NBG)], and yield itself (YLD). We used a core collection of 195 accessions which had been previously phenotyped and for which polymorphic sites had been identified in sequenced segments of the six genes. The associations between gene polymorphism and phenotypic traits were analyzed with Tassel. The percentages of admixture of each accession, estimated using 60 RFLP probes, were used as cofactors in the analyses, decreasing the proportion of false-positive tests (70%) due to population structure. The significant associations observed matched generally well the role of the enzymes encoded by the genes known to determine starch amount or type. Sh2, Bt2, Ae1, and Wx were associated with TGW. SssI and Ae1 were associated with PGT, a trait influenced by amylopectin amount. Sh2 was associated with AM while Wx was not, possibly because of the absence of waxy accessions in our collection. O2 and Wx were associated with HD and ET. No association was found between O2 and PR. These results were consistent with QTL or association data in sorghum and in orthologous zones of maize. This study represents the first targeted association mapping study for grain quality in sorghum and paves the way for marker-aided selection.

Accessing genetic diversity for crop improvement.

Vast germplasm collections are accessible but their use for crop improvement is limited-efficiently accessing genetic diversity is still a challenge. Molecular markers have clarified the structure of genetic diversity in a broad range of crops. Recent developments have made whole-genome surveys and gene-targeted surveys possible, shedding light on population dynamics and on the impact of selection during domestication. Thanks to this new precision, germplasm description has gained analytical power for resolving the genetic basis of trait variation and adaptation in crops such as major cereals, chickpea, grapevine, cacao, or banana. The challenge now is to finely characterize all the facets of plant behavior in carefully chosen materials. We suggest broadening the use of 'core reference sets' so as to facilitate material sharing within the scientific community.

Phylogeographic evidence of crop neodiversity in sorghum.

Sorghum has shown the adaptability necessary to sustain its improvement during time and geographical extension despite a genetic foundation constricted by domestication bottlenecks. Initially domesticated in the northeastern part of sub-Saharan Africa several millenia ago, sorghum quickly spread throughout Africa, and to Asia. We performed phylogeographic analysis of sequence diversity for six candidate genes for grain quality (Shrunken2, Brittle2, Soluble starch synthaseI, Waxy, Amylose extender1, and Opaque2) in a representative sample of sorghum cultivars. Haplotypes along 1-kb segments appeared little affected by recombination. Sequence similarity enabled clustering of closely related alleles and discrimination of two or three distantly related groups depending on the gene. This scheme indicated that sorghum domestication involved structured founder populations, while confirming a specific status for the guinea margaritiferum subrace. Allele rooted genealogy revealed derivation relationships by mutation or, less frequently, by recombination. Comparison of germplasm compartments revealed contrasts between genes. Sh2, Bt2, and SssI displayed a loss of diversity outside the area of origin of sorghum, whereas O2 and, to some extent, Wx and Ae1 displayed novel variation, derived from postdomestication mutations. These are likely to have been conserved under the effect of human selection, thus releasing valuable neodiversity whose extent will influence germplasm management strategies.

Construction and characterization of a Coffea canephora BAC library to study the organization of sucrose biosynthesis genes.

The first bacterial artificial chromosome (BAC) library of Robusta coffee (Coffea canephora) was constructed, with the aim of developing molecular resources to study the genome structure and evolution of this perennial crop. Clone 126, which is highly productive and confers good technological and organoleptic qualities of beverage, was chosen for development of this library. The BAC library contains 55,296 clones, with an average insert size of 135 Kb per plasmid, therefore representing theoretically nine haploid genome equivalents of C. canephora. Its validation was achieved with a set of 13 genetically anchored single-copy and 4 duplicated RFLP probes and yielded on average 9 BAC clones per probe. Screening of this BAC library was also carried out with partial cDNA probes coding for enzymes of sugar metabolism like invertases and sucrose synthase, with the aim of characterizing the organization and promoter structure of this important class of genes. It was shown that genes for both cell wall and vacuolar forms of invertases were probably unique in the Robusta genome whereas sucrose synthase was encoded by at least two genes. One of them (CcSUS1) was cloned and sequenced, showing that our BAC library is a valuable tool to rapidly identify genes of agronomic interest or linked to cup quality in C. canephora.

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.

Creation of BAC genomic resources for cocoa ( Theobroma cacao L.) for physical mapping of RGA containing BAC clones.

We have constructed and validated the first cocoa ( Theobroma cacao L.) BAC library, with the aim of developing molecular resources to study the structure and evolution of the genome of this perennial crop. This library contains 36,864 clones with an average insert size of 120 kb, representing approximately ten haploid genome equivalents. It was constructed from the genotype Scavina-6 (Sca-6), a Forastero clone highly resistant to cocoa pathogens and a parent of existing mapping populations. Validation of the BAC library was carried out with a set of 13 genetically-anchored single copy and one duplicated markers. An average of nine BAC clones per probe was identified, giving an initial experimental estimation of the genome coverage represented in the library. Screening of the library with a set of resistance gene analogues (RGAs), previously mapped in cocoa and co-localizing with QTL for resistance to Phytophthora traits, confirmed at the physical level the tight clustering of RGAs in the cocoa genome and provided the first insights into the relationships between genetic and physical distances in the cocoa genome. This library represents an available BAC resource for structural genomic studies or map-based cloning of genes corresponding to important QTLs for agronomic traits such as resistance genes to major cocoa pathogens like Phytophthora spp ( palmivora and megakarya), Crinipellis perniciosa and Moniliophthora roreri.

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.

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.

Sequence polymorphism from EST data in sugarcane: a fine analysis of 6-phosphogluconate dehydrogenase genes

O presente estudo apresenta resultados preliminares demonstrando a utilizaçäo da base de dados de ESTs de cana-de-açúcar para detectar polimorfismo de base única (SNP para Single Nucleotide Polymorphism). Sessenta e quatro ESTs relacionados aos genes da 6-phosphogluconate deshydrogenases (Pgds) foram identificados e divididos em dois conjuntos bem delimitados, de 14 e 50 ESTs, correspondendo a dois genes, A e B. O alinhamento das seqüências do grupo A permitiu a detecçäo de um único SNP e o alinhamento das seqüências do grupo B permitiu a detecçäo de 39 SNP, incluindo 27 na regiäo codificante do gene. Trinta e oito SNP foram bi-nucleotídicos e um único tri-nucleotídico. Nove inserções/supressões de um até 72 pares de base foram detectados nas regiões näo- codificantes 3' ou 5'. A robustez e as conseqüências dessas observações preliminares säo discutidas.

Genetic mapping of maize streak virus resistance from the Mascarene source. I. Resistance in line D211 and stability against different virus clones.

Maize streak virus (MSV) disease may cause significant grain yield reductions in maize in Africa. Réunion island maize germplasm is a proven source of strong resistance. Its genetic control was investigated using 123 RFLP markers in an F(2) population of D211 (resistant) × B73 (susceptible). This population of 165 F(2:3) families was carefully evaluated in Harare (Zimbabwe) and in Réunion. Artificial infestation was done with viruliferous leafhoppers. Each plant was rated weekly six times after infestation on a 1-9 scale previously adjusted by image analysis. QTL analyses were conducted for each scoring date, and for the areas under the disease, incidence and severity progress curves. The composite interval mapping method used allowed the estimation of the additive and dominance effects and QTL × environment interactions. Heritabilities ranged from 73% to 98%, increasing with time after infestation. Resistance to streak virus in D211 was provided by one region on chromosome 1, with a major effect, and four other regions on chromosomes 2, 3 (two regions) and 10, with moderate or minor effects. Overall, they explained 48-62% of the phenotypic variation for the different variables. On chromosome 3, one of the two regions seemed to be more involved in early resistance, whereas the second was detected at the latest scoring date. Other QTLs were found to be stable over time and across environments. Mild QTL × environment interactions were detected. Global gene action appeared to be partially dominant, in favor of resistance, except at the earliest scoring dates, where it was additive. From this population, 32 families were chosen, representing the whole range of susceptibility to MSV. They were tested in Réunion against three MSV clones, along with a co-inoculation of two of them. Virulence differences between clones were significant. There were genotype × clone interactions, and these were more marked for disease incidence than for severity. Although these interactions were not significant for the mean disease scores, it is suggested that breeders should select for completely resistant genotypes.

Genetic mapping of maize streak virus resistance from the Mascarene source. II. Resistance in line CIRAD390 and stability across germplasm.

The streak disease has a major effect on maize in sub-Saharan Africa. Various genetic factors for resistance to the virus have been identified and mapped in several populations; these factors derive from different sources of resistance. We have focused on the Réunion island source and have recently identified several factors in the D211 line. A second very resistant line, CIRAD390, was crossed to the same susceptible parent, B73. The linkage map comprised 124 RFLP markers, of which 79 were common with the D211×B73 map. A row-column design was used to evaluate the resistance to maize streak virus (MSV) of 191 F(2:3) families under artificial infestation at two locations: Harare (Zimbabwe) and in Réunion island. Weekly ratings of resistance were taken and disease incidence and severity calculated. QTL analyses were conducted for each scoring date and for the integration over time of the disease scores, of incidence, and of severity. Heritability estimates (71-98%) were as high as for the D211×B73 population. Eight QTLs were detected on chromosomes 1, 2, 3, 5 (two QTLs), 6, 8, and 10. The chr1-QTL explained the highest proportion of phenotypic variation, about 45%. The QTLs on chromosomes 1, 2, and 10 were located in the same chromosomal bin as QTLs for MSV resistance in the D211×B73 population. In a simultaneous fit, QTLs explained together 43-67% of the phenotypic variation. The QTLs on chromosomes 3, 5, and 6 appeared to be specific for one or the other component of the resistance. For the chr3-QTL, resistance was contributed by the susceptible parent. There were significant QTL × environment interactions for some of the variables studied, but QTLs were stable in the two environments. They also appeared to be stable over time. Global gene action ranged from partial dominance to overdominance, except for disease severity. Some additional putative QTLs were also detected. The major QTL on chromosome 1 seemed to be common to the other sources of resistance, namely Tzi4, a tolerant line from IITA, and CML202 from CIMMYT. However, the distribution of the other QTLs within the genome revealed differences in Réunion germplasm and across these other resistance sources. This diversity is of great importance when considering the durability of the resistance.

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.

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.