Molecular markers for DNA-fingerprinting in cotton

Molecular markers are long proposed as suitable for fingerprinting of crop species. With the development of the techniques the attention has shifted from protein to DNA-based markers as age-, tissue- and status-independent and, therefore, more reliable and reproducible. Several such markers (RFLP, RAPD, AFLP, SSR) were used in cotton for developing a high density genetic map for genomic studies and with the prospect for marker-assisted selection. The availability of these markers, together with the existing possibility for genetic transformation of the crop, make the development of DNA .fingerprints. for the varieties an issue of rising interest among breeders and seed companies. To the best of our knowledge no concentrated effort for developing such fingerprints has been made up to now in spite of the several reports proposing the potential usefulness of microsatellites and AFLPs in this respect. Here we present our work on comparing applicability of three types of molecular markers (microsatellites, AFLPs and SAMPLs) towards establishing a reliable, reproducible and relatively inexpensive protocol for DNA-fingerprinting for cotton. Furthermore, the intended transferability to the widest possible range of laboratories and, therefore, bringing it the closest to the breeder.s fields, was attempted through the replacement of the standard radioactive DNA labeling procedure with the less hazardous .silver staining . protocol. The outcomes and the possible extensions of the present work are discussed. (Résumé d'auteur

Genetical genomics dissection of cotton fiber quality

Cotton fiber is a commodity of key economic importance in both developed and developing countries. The two cultivated species, Gossypium hirsutum and G. barbadense , are tetraploid (2n=1x= 52 . 2.3 Gb). Cotton fibers are single-celled trichomes of the outermost epidermallayer of the ovule and elongate extensively to 25-50 mm. The final quality of the fiber results from complex developmentai processes and improvement of cotton fiber quality remains a challenge for many research groups worldwide. Although traditional breeding methods have proven efficient, the contribution of molecular genetics and genomic tools are gaining interest and the cotton fiber transcriptome has attracted a lot of attention in recent years. The major objective of the project (acronym Cotton_RILs) sponsored by the French National Research Agency (ANR) , is the genetic and genomic dissection of important fiber quality characteristics using a combination of classical QTL mapping and of gene expression QTL mapping. The integrated genetics and genomics approach (or genetical genomics approach) in this project is centered on a population of interspecific G. hirsutum X G. harhadense recombinant inbred lines (RILs) created by CIRAD. Specifie objectives are, 1. Construction of a saturated genetic map, 2. QTL mapping through multi-site phenotypic evaluation on 1 continents. 3. Population-wide gene expression analysis through microarray and cDNA-AFLP profilings and for 1 or 2 key developmental stages, and 4. Genetic fine mapping of selected QTLs using a large F, population. The 3 participants in the project. CIRAD (Montpellier, France) . Bayer Crop Science (Gent . Belgium). and CSIRü (Canberra , Australia) , have active research programs in applied genetics both through c1assical breeding and using modern biotechnology. Past achievements of the 3 laboratories are recognized worldwide and they are highly complementary in terms of their scientific expertise. Apart from greenhouses and biotechnology laboratories in their respective primary sites, they provide access to a broad range of field experimental sites on 4 continents, in Brazil and Cameroon through CIRAD partnerships and in the USA for Bayer CS. (Texte intégral

Combining genetic and genomics approaches for fiber quality improvement in tetraploid cotton

Cotton fibers are the premier natural fibers for textile production. The two tetraploid species, Gossypium barbadense and G. hirsutum, differ significantly in their fiber properties, the former having much longer, finer and stronger fibers. A better understanding of the genetics and underlying biological causes of these differences will aid further improvement of cotton quality through breeding and biotechnology. In the recent period, the availability of a genetically stable interspecific RIL population (140 F2:8 individuals) allowed CIRAD, Bayer Crop Science and CSIRO, to benefit from the financial support of the French National Research Agency (ANR) for a project named "genetic and genomic dissection of cotton fiber quality". The genetic map of the RILs (800 loci, 2044 cM) and more importantly the consensus map resulting from its integration with the BC1 map (75 BC1 + 140 RILs, 1745 loci, 3637 cM) now constitutes one of the best basis internationally, as it offers a maximized number of bridge loci in common with other interspecific G hirsutum x G barbadense maps (TAG, 2009, 119:281-292). The phenotypic characterisation of the fibers of the RILs on 4 continents and several growing seasons generated 11 independent fiber data sets which served for QTL mapping. The 167 significant fiber QTLs (LOD>permutation based threshold) and 651 putative LOD peaks (LOD>2) from these RIL experiments were integrated with QTLs from the BC1 and from the literature. The meta-analysis of this large set of QTL data using MetaQTL software indicated that some chromosome regions hosted "confirmed" QTLs. An effective co-localization of unidirectional (similar sign of additivity) LOD peaks from at least 5 independent data sets was observed in at least 26 cases (a given fiber trait and a given chromosome region) where meta-clusters of QTLs were defined (BMC Plant Biol, 2010, 10:132). The fiber trancriptomes of the 2 parents and of a set of 88 RILs were analyzed with focus on 2 key developmental stages (10 and 22 days post anthesis) using 2 profiling techniques, quantitative 3' targeting cDNA-AFLP and microarray hybridizations. Both platforms showed their utility for the population-wide profiling of the differential expression of an important number of gene transcripts (4,400 and 22,000 respectively). QTL analysis applied to gene expression resulted in large numbers of (>5000) expression QTLs. This is the first report of an application of a genetical genomics approach in cotton. (Texte intégral

Microsatellite allelic diversity within tetraploid Gossypium germplasm

For cotton like for most important crops, future improvements of adaptation to adverse environment, of agronomic fitness and of quality of agricultural products, are expected to rely on the better utilization of genetic resources. Opportunities for such utilization are offered by the techniques of molecular biology through molecular breeding and genetic transformation. Within the major tetraploid cultivated species, G. hirsutum, two pools can be distinguished as sources of genetic variability for cotton breeders: the cultivated pool and the primitive, or exotic, pool. It is recognized that a severe bottleneck has accompanied the domestication process leading to modern commercially important cotton cultivars, resulting in a reduced genetic base. On another hand, the exotic tetraploid germplasm pool is constituted of landraces (7 geographical races belong to the G. hirsutum sp.), feral accessions and wild tetraploids, most being perennial and photoperiodic shrubs. This important gene reservoir, though constituted of morphologically and ecologically diverse germplasm, has received minor attention. Studies using isozymes and RFLP markers have early shown the limited diversity in the cultivated pool of G. hirsutum sp, thus explaining the choice made by many teams to initiate in the early 90s essentially interspecific marker-assisted breeding programs. The development of more efficient marker-systems, like AFLPs, and the availability of an increasing number of sources of microsatellites (SSRs) in the public (ca 1000) and private (min 2000) has now changed the vision. We used different sources of SSRs to assess the molecular polymorphism of a set of G. hirsutum cultivars and races, as well as of exotic tetraploid accessions. The 2 primary sources of SSRs belong to the "BNL" and "CIR" series, all chosen after placement of corresponding loci on our genetic map (Nguyen et al. TAG, 2004, 109:167-175). The plant material comprised a collection of 48 accessions, chosen in our gene bank and representing the variability of G. hirsutum-related landraces (7 geographical races and Moco cultigens) and of 3 other tetraploid species, G. barbadense, G. tomentosum and G. darwinii. This material was analysed for allelic diversity with 135 SSRs (236 SSRs analysed in a first run), which corresponded to 147 different loci. In total, 818 alleles were detected, varying between 2 and 17 alleles per locus, with an average of 5.6 alleles. The genetic distances calculated from the allelic data structured the genetic diversity in accordance with the known botanical, genealogical and/or geographical information, and confirmed previous molecular phylogenetic trees. The relative informativeness of SSRs assessed by the number of alleles or by the Nei statistics varied considerably. A "genotyping kit" was assembled based upon the most informative SSRs, that were re-associated in multiplexes by three (3-plexes are routinely used in our lab): 2 to 3 triplexes were assembled for each of the 13 pairs of homoeologous chromosomes of the tetraploid cotton genome. The applications of such a genome-wide genotyping kit of SSR markers in cotton are very important for phylogenetics, fingerprinting of cotton cultivars, or germplasm collection management. (Résumé d'auteur

Improvement of cotton fiber quality through marker-assisted breeding: limits and prospects of a QTL approach

The marker-assisted introgression of quantitative traits in crop plants is the topic of an ever-increasing number of reports (recent interrogation of bibliographic database using marker-assisted breeding or QTL as key words produced nearly 500 and 2500 hits respectively). Different papers based both on theoretical and experimental evidence, have also emphasized the limits of QTL-based breeding strategies when applied to quantitative traits. Among these limitations the molecular breeder may face are the precision, power and confidence associated to QTL detection, the important number of progenies to be manipulated when the number of QTL increases. A combination of marker-based and classical phenotype-based selections is often recommended. Our program of interspecific G. hirsutum / G. barbadense marker-assisted backcross selection, MABS, aims at transferring cotton fibre quality QTLs of G. barbadense into G. hirsutum. In the preceding seasons, the BC1, BC2 and BC2S1 served for 3 separate QTL analyses (Lacape et al., Crop Sc, in press). The pooled QTL data from the 3 populations revealed a total of 50 QTLs meeting permutation-based LOD thresholds (between LOD 3.2 and LOD 4.0 on a per-trait basis) for 11 fibre quality-related traits. When reducing the detection threshold to LOD 2.5, this total reached 80 QTLs. Individual phenotypic effects were essentially in the range of 5-15%, and rarely exceeded 20%. The direction of the effect of 70% of the QTLs was as expected from the parental value (better fibre length, strength and fineness conferred by the G. barbadense parent, and better maturity and colour conferred by the G. hirsutum parent alleles). A majority of the QTLs were co-localized within QTL-rich chromosome regions: when considering only the donor segments, for which positive contributions to fibre quality derived from the presence of the G. barbadense alleles, 19 regions on 15 different chromosomes were defined. Interestingly, 1/3rd (26 out of 80) of the QTLs (LOD 2.5) detected confirmed QTLs reported in other interspecific populations. Using these 15 regions as target candidate regions for introgression, we undertook marker-based selection within 411 and 450 plants in each BC3 and BC4 generations respectively: ca 10% of individuals were kept at seedling stage based on the presence of G. barbadense alleles at chosen SSR and AFLP loci. The allelic constitution of 37 selected BC3 plants showed that 5 to 16% (8% on average) of donor G. barbadense alleles were still present genome-wide, corresponding only to 3 to 6 of the 15 initial QTL-rich target regions. The marker-trait associations in this BC4 generation only partly confirmed the associations observed in the previous generations (around cases). Some of the BC4 plants having introgressed these particular regions however proved highly interesting as regards to their fibre quality. The field evaluation of these BC4 as F2 progenies value using agronomical as well as fibre quality measurements was underway during summer 2004. (Résumé d'auteur

Major results and research challenges in cotton molecular genetics at Cirad (France)

CIRAD (Montpellier, France) develops research activities centered on tropical and sub-tropical agricultural systems. Among others crops, cotton is the fucus of a series of research programs in different disciplines from economics to breeding. Major areas in genetics and breeding relate to (1) genetic diversity, (2) cultivar development through classical and molecular breeding, and (3) applied genomics. An important but under-exploited reservoir of genetic diversity exists within the genus Gossypium. With over 3000 accessions CIRAD has one of the few important gene banks worldwide. It includes cultivars, landraces , and wild Gossypiurn species. DNA molecular markeTs have shawn their usefulness and validity for fingerprinting plant resources and for structuring diversity of large collections. In our case, after a preliminary step centered on the screening of microsatellite markers over a limited set of accessions of the gene bank (a genotyping kit has been proposed) , our future plans include the use of high-throughput methods of genotyping (a DArT platform will soon be available in our unit). Opportunities for exploiting this large diversity relate to the development core-collections and of genetic association studies for a given set of traits (fiber quality, stress response) . After a past and fruitful period of collaboration in different tropical countries (varieties co-obtained by ClRAD are cultivated on over 2. 5 million hectares worldwide), CIRAD has more recently engaged research programs in molecular genetics. Focus has initially been on fiber quality as a target trait and on markerassisted interspecific breeding for introgressing fiber quality traits from G. barbadense into G. hirsutUtn. Achievements in this area (genetic maps, fiber quality QTLs) have been published and are posted on the web. The database of CIRAD, TropGene DB (available at http, lltropgenedb. cirad. h l ) is regularly updated and presently displays various genetic maps as well as QTL data. Based upon QTL detected in the BCI and BC2 (backcross to the Gh parent) generations, marker-assisted selection in further generations (BC3 to BCG) was conducted and advanced lines introgressed with 3 ~ 5 QTLs were tested under tropical conditions. Using the same material, libraries of segmental introgression lines are being created for a few specifie target chromosomes. More recently , through collaboration with Embrapa (Brazil), CIRAD has initiated research on disease and nematode resistances in order to clarify the genetic basis of resistance; prepare segregating populations, and develop marker-assisted selection programs. A new collaborative project coordinated by CIRAD on the genetics and genomics of cotton fiber quality has been launched in 2007. CIRAD and its partners, Bayer CS and CSIRü, are engaged in ambitious research aimed at elucidating the genetic basis of fiber quality. The project is centered on an interspecific RIL population (a cross between 2 weil studied parents , Guazuncho 2 (G. hirsuturn) and VH8 (G. harhadense)) . It is innovative by the approach combining (1) phenotypic assessment of fi ber quality from field and greenhouse experiments on four continents, and (2) transcriptome profiling using complementary platforms, hybridization-based (microarrays) and PCR-based (cDNA-AFLP). Comparison of QTL and eQTL results is expected to help in identifying candidate genes important in fiber quality. (Résumé d'auteur

Genome mapping of tetraploid cotton : towards a saturated and unified map

DNA-based genetic maps have been produced in nearly all major crop species, thus facilitating the analysis of genome structure and evolution, and improving efficiency and accuracy of breeding. We have developed at Cirad/France a combined and saturated RFLP-AFLP-SSR genetic map of tetraplaid cotton from the analysis of the 1st and 2nd backcross generations of an interspecific Gossypium hirsutum (cv 'Guazuncho 2') x G. barbadense (cv 'VH8') cross. The BC1 and BC2 maps were independently constructed from the analysis of 75 arid 200 individual plants respectively. As a recent development, a microsatelliteenriched library had been developed and 418 new microsatellite primers defined. One hundred and sixty one microsatellites showing at least one polymorphism between Guazuncho 2 and VH8 had been screened on the BC1 population, and 785 new loci were added on the BC 7 map. Having 360 loci in common between the BC1 (1107 loci in total) and tile BC2 (513 loci in total) map proved helpful to confirm loci orders along linkage groups, and allowed small linkage groups to be joined to larger ones. A total of 138 additional loci, mainly AFLP, of the BC2 map were added to the skeleton BC? map. Finally, after merging the BC1 and BC2 mapping data, the Guazuncho 2 x VH8 map now comprises 1260 loci divided mainly between AFLPs (40%), SSRs (34%) and RFLPs (15%). The 26 linkage groups span altogether over 5400 cM. Details of the Guazuncho 2 x VH8 map are commented. An important number of microsatellite and RFLP markers used in the present study are common with other published genetic maps also derived from interspecific G, hirsutum x G. barbadense populations. As a preliminary step towards an integration of the different maps, we used a "neighbor approach" applied to 4 of tth 26 chromosomes by aligning these maps using these bridge loci. An overall agreement in locus order and distances has been observed. Integration of genetic maps into a denser consensus unified map of tetraploid cotton and future feasibility of genetic arid physical maps integration are discussed. (Résumé d'auteur

Study of the Determinism of the glanded-plant and glandless-seed trait introgressed in G. hirsutum from G. sturtianum

Two hundred and six mapped microsatellites markers evenly distributed on the 26 chromosomes of Gossypium hirsutum L. were used to monitor the introgression of DNA fragments coming from the Australian species G. sturtianum Willis and the wild American diploid species G. ramondii Ulb. in a population of BC1, BC2, BC2S1, BC2S2, BC2S3, BC2S4, BC2S5, BC2S2/BC1, BC2S2/BC1/S1,BC3, BC3S1, BC3S2 and BC3S3 derivatives obtained from the G. hirsutum x G. raimondii x G. sturtianum (HRS) trispecific hybrid. In the most advanced backcrossed progenies of the HRS hybrid, the only plants that are still showing a drastic inhibition of gossypol synthesis in a part of their seeds and a normal glanding pattern in their other organs contain fragments of G. sturtianum DNA related to c02- c1, c03-c17 and c06-c025 linkage groups of G. hirsutum. In these plants, all the SSR markers associated to the G. sturtianum c06-c025 DNA fragment introgressed in G. hirsutum remain heterozygous after numerous generations of selfing. One can thus suppose that this alien chromosomic fragment may also carry a recessive lethal factor, which expresses itself when it becomes homozygous. If this hypothesis proves to be true, it will be necessary to break the linkage that exist between this lethal factor and the gene(s) responsible for the expression of the trait of interest in order to develop stable homozygous cotton lines with very low gossypol content in the seed and high gossypol content in the aerial parts. (Résumé d'auteur

eQTL analysis from microarray data of fibre expressed genes in an inter-specific Gossypium hirsutum x G. barbadense RIL population.

Microarrays provide a wealth of genome-wide gene expression data to characterise the biological variability between different plant genotypes, however it remains difficult to link such genomic results to a functional interpretation of how specific gene action determines a particular plant phenotype. Phenotypes themselves can be associated with specific regions of the genome by traditional QTL mapping, but most QTLs are very large physical regions that do not often allow the identification of specific underlying genes, particularly in species like cotton lacking a full genome sequence. Combining QTL and microarray analyses is a novel way to narrow down on subsets of candidate genes whose level of expression can be statistically correlated to regions of the genome, so-called eQTLs, and further correlated to specific phenotypes. In this study we used a RIL population of 145 lines from an inter-specific cross between a G. barbadense Sea-Island accession (VH8) with very high fibre quality and a G. hirsutum cultivar of moderate fibre quality (Guazuncho II). The RIL population had been phenotyped for fibre quality traits at multiple sites and in most cases over multiple years and had also been genotyped. The consensus genetic map for the population contained over 600 SSR and AFLP markers and was used as the framework for both phenotypic QTL and eQTL mapping. Microarray analyses (approx. 24,000 genes per array) were carried out on 10 dpa fibre cDNA on 102 of the RILs and the individual gene expression values for each transcript treated as a quantitative trait and mapped to the genome. The distribution of eQTLs was not uniform across the chromosomes, with chromosomes 5 and 12 having more eQTLs than any others. A number eQTL hotspots were identified that may represent the locations of master regulators of fibre expressed genes. A selection of genes co-located with the phenotypic QTLs for fibre traits were also identified and validated by quantitative PCR. Some, but not all eQTLs identified from the microarray data were confirmed by Q-PCR. Large correlations tables of the expression of all the genes on the arrays and specific fibre traits like length, strength and fineness were developed and are also being used to identify potential candidate genes that may be causal in conferring the commercially useful properties of cotton fibres. (Résumé d'auteur