Transferability of the EST-SSRs developed on Nules clementine (Citrus clementina Hort ex Tan) to other Citrus species and their effectiveness for genetic mapping

Franois L Luro 2 Gilles Costantino 2 Javier Terol 1 Xavier Argout 0 Thierry Allario 4 Patrick Wincker 3 Manuel Talon 0 Patrick Ollitrault 4 Raphael Morillon 4 0 CIRAD AMIS , Montpellier , France 1 Centro de Genomica, Instituto Valenciano de Investigationes Agrarias , Valencia , Spain 2 INRA, Unite de Recherche GEQA, INRA San Giuliano , 20230 San Nicolao , France 3 Genoscope, CNS , Evry , France 4 UPR 'Amelioration genetique d'especes a multiplication vegetative' , CIRAD, Montpellier , France Background: During the last decade, numerous microsatellite markers were developed for genotyping and to identify closely related plant genotypes. In citrus, previously developed microsatellite markers were arisen from genomic libraries and more often located in non coding DNA sequences. To optimize the use of these EST-SSRs as genetic markers in genome mapping programs and citrus systematic analysis, we have investigated their polymorphism related to the type (di or trinucleotide) or their position in the coding sequences. Results: Among 11000 unigenes from a Clementine EST library, we have found at least one microsatellite sequence (repeated units size ranged from 2 to 6 nucleotides) in 1500 unigenes (13.6%). More than 95% of these SSRs were di or trinucleotides. If trinucleotide microsatellites were encountered trough all part of EST sequences, dinucleotide microsatellites were preferentially (50%) concentrated in the 5' 100th nucleotides. We assessed the polymorphism of 41 EST-SSR, by PCR amplification droved with flanking primers among ten Citrus species plus 3 from other genera. More than 90% of EST-SSR markers were polymorphic. Furthermore, dinucleotide microsatellite markers were more polymorphic than trinucleotide ones, probably related to their distribution that was more often located in the 5' UnTranslated Region (UTR). We obtained a good agreement of diversity relationships between the citrus species and relatives assessed with EST-SSR markers with the established taxonomy and phylogeny. To end, the heterozygosity of each genotype and all dual combinations were studied to evaluate the percentage of mappable markers. Higher values (> 45%) were observed for putative Citrus inter-specific hybrids (lime lemon, or sour orange) than for Citrus basic true species (mandarin, pummelo and citron) (<30%). Most favorable combinations for genome mapping were observed in those involving interspecific hybrid genotypes. Those gave higher levels of mappable markers (>70%) with a significant proportion suitable for synteny analysis. Conclusion: Fourty one new EST-SSR markers were produced and were available for citrus genetic studies. Whatever the position of the SSR in the ESTs the EST-SSR markers we developed are powerful to investigate genetic diversity and genome mapping in citrus. - Background Simple Sequence Repeats are tandem repeat sequences that are quite abundant in eukaryotes genomes [1]. Numerous genomic libraries enriched in SSR have been established from many plant species [2-5]. Those repeat sequences also called microsatellites (MS) present a higher level of polymorphism and higher expected heterozygosity when compared with to other dominant (AFLP and RAPD) or codominant markers (RFLP) [6]. Since SSRs are ubiquitously present in genomes with randomly occurrence, they are communally used as genetic markers in many different plant species to unravel the interspecific and intraspecific diversity [7-10]. In citrus, the number of published markers of genomic SSRs is still limited [11,12]. Those markers were used for genetic diversity assessment and for germplasm management [13,14]. A high-density microsatellite consensus map is still lacking. The major goal of genetic mapping is to localize genes or QTLs, involved in traits of interest that are linked to molecular markers. Those molecular markers can be used as a starting point for gene identification or to reduce schemes of selection. One other way to address this aim is to develop markers directly localized in the coding sequences. ESTs (Expressed Sequence Tags) derived from cDNA libraries obtained from the genome expression have been investigated for microsatellite screening, in barley [15], wheat [16], rice [17], citrus [18,19], sugarcane [20] and grape [21]. It is assumed that those SSRs markers should enable to assess the molecular evolution of the genes in which they are positioned. Indeed, it has been observed that in ESTs, the flanking region of SSRs are more conserved and can also be found in related genera [22]. Thousands of EST-SSRs were identified in numerous species such as grape and cereal. A high level of transferability was noted between rice, wheat and barley [17]. In citrus, thousands of ESTs are now available in databases. Recently, using public sequence databases resources, Chen et al. [23], published the characterization of 56 EST-SSR markers identified among 2295 citrus ESTs, mappable in a progeny obtained from a cross between sweet orange (Citrus sinensis L. Osb.) and trifoliate orange (Poncirus trifoliata L. Raf.). If those two genotypes represent important resources of agronomical characters for rootstock and cultivar improvement scheme, numerous other citrus species offer a large panel of specific traits interesting breeders or consumers. For example, Clementine (Citrus clementina Hort. Ex Tan.) is a model citrus crop in Mediterranean area and sour orange (C. aurantium L.) or Cleopatra mandarin (C. reshni Hort. Ex Tan.) are tolerant to abiotic constraints such as salt stress or calcareous soils [24]. Citrus as many fruit trees have a juvenility period with around 5 years of duration limiting the possibility to study the allelic segregation on a second generation of hybrids (F2 or BC). Consequently citrus genetic maps are established on F1 progenies at interspecific [25], and intergeneric levels [26-31]. To maximize the potential for the development of EST-SSR based maps we need to investigate the polymorphism and the heterozygosity of these markers in different combined genotypes at the origin of F1 progenies. Another point of reflexion concerning the polymorphism of SSRs in EST is the usefulness of the derived markers such as STMS (Sequence Tagged MicroSatellite) in cultivar distinctness and in relationships between varieties and species. The particular position of these SSRs inside coding sequences may question the genetic diversity information that we can extract from those markers related to the putative influence of the selection on the SSR polymorphism. In a full-length clementine (Citrus clementina) ESTs database [19], we looked for SSR markers. Screening of 37 000 ESTs allowed us to identify about 1600 SSRs. We report here the outline investigation of the polymorphism of EST-SSR among a set of 16 citrus species covering a wide range of citrus genetic diversity. We assessed also the mappability of these markers on our different progenies established for heredity studies. The e (...truncated)