Pierre Lagoda

Molecular breeding in the genus Musa: a strong case for STMS marker technology

Musa species are among the tallest monocotyledons and include major food-producing species. The principal cultivars, derived from two major species Musa acuminata (‘A’ genome) and Musa balbisiana (‘B’ genome), are polyploid hybrids (mainly AAA, AAB and ABB triploids), medium to highly sterile, parthenocarpic and clonally propagated. Bananas and plantains are crops to which molecular breeding is expected to have a positive impact. In order to better understand banana genetics, more knowledge has to be accumulated about the complex genome structure of hybrids and cultivars. Therefore, the aim of our work is to develop molecular markers that are codominant, reliable, universal, highly polymorphic and that are applicable to collaborative Musa germplasm genotyping and mapping. Two size-selected genomic libraries have been screened for the presence of simple sequence repeats (SSR). Our data demonstrate that SSR are readily applicable to the study of Musa genetics. Our comprehensive analyses of a significant number of banana sequence tagged microsatellite sites (STMS) will add to our knowledge on the structure and phylogeny of genomes of the Musa species, and suggest that microsatellites be used as anchor markers for a banana genetic core map. Additional markers, such as e.g. CAPS have also been tested in order to increase the detection of polymorphisms exceeding that revealed by STMS technology. The utility of PCR-derived markers for collaborative genetic analyses of the banana genome, and the transferability of streamlined’ laboratory techniques and data analysis to Developing Countries are discussed.

Rice genomics: Present and future

A review of the present and future of rice genomics is presented. Rice is a model species for cereals as well as a very important crop. Its genome has been the focus of many mapping experiments associated with QTL localization. These genetic maps now serve as a background for physical mapping, genome sequencing and gene discovery. Recent progress are reviewed. The next step in rice genomics is functional genomics with the determination of the function of the genes. The most straightforward approaches are discussed.

Diploid ancestors of triploid export banana cultivars: molecular identification of 2n restitution gamete donors and n gamete donors

The origin of triploid export banana cultivars was investigated. They all belong to Cavendish and Gros Michel subgroups of triploid clones and have a monospecific Musa acuminata origin. The appearance of these cultivars is thought to be result of hybridization between partially sterile diploid cultivars producing non reduced gametes and fertile diploids producing normal haploid gametes. To trace these diploid ancestors we compared the RFLP patterns, revealed by 36 probe/enzyme combinations, of 176 diploid clones representing the worldwide available variability with that of clones from the Cavendish and Gros Michel subgroups. This lead us to the identification of the common putative diploid ancestor of cultivars from Cavendish and Gros Michel subgroups which contributed to triploid cultivar formation through the production of 2n restitution gametes. For cultivars of Gros Michel subgroup we also propose a normal gamete donor that may have complemented the triploid allele set.

DNA libraries for sequencing the genome of Ostreococcus tauri (Chlorophyta, prasinophyceae): The smallest free-living eukaryotic cell

Ostreococcus tauri is a marine photosynthetic picoeukaryote presenting a minimal cellular organization with one nucleus, one chloroplast, and one mitochondrion. It has the smallest genome described among free‐living eukaryotic cells, and we showed by pulsed‐field gel electrophoresis (PFGE) that it is divided between 15 bands ranging from 1.2 to 0.15 Mb, giving a total size of 9.7 Mb. A Bacterial Artificial Chromosome (BAC) library was prepared from genomic DNA extracted from a culture of O. tauri. A total of 2457 clones was obtained with an average insert size of around 70 kb, representing an 18‐fold coverage of the genome. The library was spotted on high density filters, and several probes of coding sequences were hybridized to both the high density BAC library filters and directly to the dried PFGE gels of the O. tauri genomic DNA. These hybridizations allowed a preliminary organization of the library and the localization of several markers on the chromosomes. Randomly selected fragments were also sequenced, representing 12% of the O. tauri genome. Many sequences showed significant similarities in data banks, mainly with plant and algae sequences. About 1000 coding sequences could be identified. These data confirmed the position of O. tauri in the green lineage and the hypothesis of a very compact organization of its genome.

A new aspect of genetic diversity of Indonesian oil palm (Elaeis guineensis Jacq.) revealed by isoenzyme and AFLP markers and its consequences for breeding

Abstract Oil palm (Elaeis guineensis Jacq.) plays an important economic role in some countries of Southeast Asia like Indonesia, which is the world’s second producer of palm and palm kernel oil. The quality improvement of planting material needs a better understanding of the genetic relationships between genotypes from different populations used in the breeding programmes. In this study, 48 parents, representative of four populations used in Indonesian Oil Palm Research Institute (IOPRI) breeding programmes, were analysed with five selected AFLP primer pairs and four isoenzymatic systems. One hundred and fifty eight scorable band levels were generated of which 96 (61%) were polymorphic. AFLP allowed us to identify off–type descendants which were excluded from analysis. The use of unbiased Rogers distance clearly separated the four studied populations. The Neighbor-Joining method re-groups two African populations which are known as originating from different regions. Nevertheless, the variability revealed is in accordance with oil palm breeders’ knowledge. The results obtained with AFLP showed that the crosses among the African sub-population, which is excluded in oil palm reciprocal recurrent selection (RRS) breeding programmes, may be more interesting than the crosses between the African and the Deli populations.

TropGENE‐DB, a multi‐tropical crop information system

TropGENE-DB, is a crop information system created to store genetic, molecular and phenotypic data of the numerous yet poorly documented tropical crop species. The most common data stored in TropGENE-DB are information on genetic resources (agro-morphological data, parentages, allelic diversity), molecular markers, genetic maps, results of quantitative trait loci analyses, data from physical mapping, sequences, genes, as well as the corresponding references. TropGENE-DB is organized on a crop basis with currently three running modules (sugarcane, cocoa and banana), with plans to create additional modules for rice, cotton, oil palm, coconut, rubber tree, pineapple, taro, yam and sorghum. The TropGENE-DB information system is accessible for consultation via the internet at http://tropgenedb.cirad.fr. Specific web consultation interfaces have been designed to allow quick consultations as well as complex queries.

Genomics of Banana and Plantain (Musa spp.), Major Staple Crops in the Tropics

This chapter on Musa (banana and plantain) genomics covers the latest information on activities and resources developed by the Global Musa Genomics Consortium. Section 4.1 describes the morphology of the plant, its socio-economical importance and usefulness as an experimental organism. Section 4.2 describes the complexity of Musa taxonomy and the importance of genetic diversity. Section 4.3 details the genetic maps which have recently been developed and those that are currently being developed. Section 4.4 presents the five BAC libraries which are now publicly available from the Musa Genome Resource Centre and can be distributed in various forms under a material transfer agreement. Section 4.5 gives an overview of cytogenetics and genome organization, showing that the genus Musa has a quite high proportion of repetitive DNA; the discovery of the first para-retrovirus integrated in the genome makes it unique. Section 4.6 explains the first attempts to sequence the genome by BAC end sequencing, whole BAC sequencing, and reduced representation sequencing. Section 4.7 addresses functional genomics with the description of cDNA libraries, gene validation using gene trapping, mutation induction and tilling techniques, as well as genetic transformation. Section 4.8 draws overall conclusions. This chapter demonstrates that by organizing the Global Musa Genomics Consortium (currently comprising 33 member institutions from 23 countries), duplication of effort can be minimized and the results of Musa genomics research are rapidly made accessible to taxonomists, breeders and the biotechnology community.

Comparative mapping of the wheat 5B short chromosome arm distal region with rice, relative to a crossability locus

Abstract.Colinearity between wheat and rice genomes is quite well established at the chromosome level, but less is known at a finer level. We tried to specify these relationships for the wheat 5BS chromosome-arm distal region, where a major locus for crossability was located. By developing AFLP markers, we succeeded to locate this major QTL more precisely. One cloned AFLP fragment mapped to rice chromosome 11, which was in agreement with a rice chromosome-11 linkage block reported in this region. However a second marker, a RFLP probe, showed a break in synteny because it mapped to rice long-arm chromosomes 1 and 5, while screening a rice BAC library with the same probe identified rice chromosomes 5 and 6. Therefore, we concluded that the syntenic relationships were more complex at the fine level. The observed results might indicate the presence of a linkage block carrying a crossability gene on wheat groups 1, 5 and 7, and also on rice chromosomes 5 and 6.

Use of competitive PCR to assay copy number of repetitive elements in banana

Abstract Banana is one of the most important sub- tropical fruit crops. Genetic improvement by traditional breeding strategies is difficult and better knowledge of genomic structure is needed. Repeated sequences are powerful markers for genetic fingerprinting. The method proposed here to determine the copy number of nuclear repetitive elements is based on competitive reverse transcription-polymerase chain reaction and can also be used for quantifying cytosolic sequences. The reliability of this method was investigated on crude preparations of total DNA. Variations due to the heterogeneity of crude DNA extracts showed that a single locus reference is needed for accurate quantification. A mapped microsatellite locus was used to normalize copy number measurements. Copy number assay of repetitive elements using this method clearly distinguishes between the two banana subspecies investigated: Musa acuminata spp. banskii and M. acuminata spp. malaccensis. Two repetitive sequence families, pMaCIR1115 and pA9-26, were assayed that cover up to 1% of the M. acuminata genome. Their copy number varied up to six fold between the two subspecies. Furthermore, sequence quantification showed that mitochondrial genomes are present in crude leaf-extracted banana DNA at up to 40 copies per cell.

Inter-Alu PCR like genomic profiling in banana

Alu sequences are a major repetitive element of the primate genome. To date Alu sequences have seldom been reported in plant genomes. We report here an inter-Alu PCR like genomic profiling in banana using a single primer designed from a human Alu sequence. The different Musa species involved in the complex genome of banana cultivars can thus be discriminated. The use of this technique for monitoring germplasm or genotyping cultivars is discussed.