In silico Discovery of EST-Based SNP Markers Useful for Identification of Citrus Species and Cultivars

Sweet orange [Citrus sinensis (L.) Osbeck] represents the most important Citrus species cropped in the world followed by Clementine (C. clementina Hort. ex Tan.) in Mediterranean areas. Citrus species and genotypes are difficult to recognize as they have moderate level of diversity due to nucellar selection, vegetative propagation and the origin by single spontaneous mutation. Despite the great number of sequences available and the draft assembly of sweet orange and clementine, there are not currently SNPs databases to be exploited for genotyping. For this purpose, 540 thousands ESTs of 19 Citrus species were assembled in about 52 thousands contigs. The QualitySNP pipeline was applied to discover potential and reliable SNPs using a stringent cleaning procedure.The vast majority of ESTs, contigs and SNPs were found in C. clementina and C. sinensis: 4,400 out of 16,000 contigs (27%) of C. clementina and 4,100 out of 17,000 contigs (24%) of C. sinensis contained putative SNPs. The percentage of reliable over the total number of potential SNPs was 25% in clementine and 16% in sweet orange. A total of 3,634 sequences were associated to enzymes belonging to 121 metabolic KEGG pathways, of which 2,090 in C. sinensis and 1,544 in C. clementina. In both species, the highest number of EST sequences belonged to secondary metabolite pathway, followed by the biosynthesis of plant hormones in C. sinensis, and the phenylpropanoid biosynthesis in C. clementina. A total of 163 SNPs from 52 contigs and genes of specific functional categories were validated. From the genotyping analysis, 78 polymorphic sites were found, 36 of which were heterozygous. The SNP variations were found mainly between species, while poor within species variability was discovered. Furthermore, 37 selected SNPs, validated by Sanger sequencing, confirmed in most of the cases the genotyping result. This work provides a collection of 15,789 putative SNP markers to be useful for nucleotide diversity analysis and genetic mapping in Citrus species. The polymorphism identified through the validated SNPs, and associated to specific genes, constitute a database to be used for functional genetic studies in germplasm diversity analysis and in mapping.