Numerous molecular polymorphisms (SNPs and indels), which are potentially useful for the analysis of interspecific genetic structures, have been identified. The nuclear phylogenetic network for Citrus and its sexually compatible relatives was consistent with the geographical origins of these genera. The positive selection observed for a few genes will help further works to analyse the molecular basis of the variability of the associated traits. This study presents new insights into the origin of C. sinensis.

Most cultivated Citrus species originated from interspecific hybridisation between four ancestral taxa (C. reticulata, C. maxima, C. medica, and C. micrantha) with limited further interspecific recombination due to vegetative propagation. This evolution resulted in admixture genomes with frequent interspecific heterozygosity. Moreover, a major part of the phenotypic diversity of edible citrus results from the initial differentiation between these taxa. Deciphering the phylogenomic structure of citrus germplasm is therefore essential for an efficient utilization of citrus biodiversity in breeding schemes. The objective of this work was to develop a set of species-diagnostic single nucleotide polymorphism (SNP) markers for the four Citrus ancestral taxa covering the nine chromosomes, and to use these markers to infer the phylogenomic structure of secondary species and modern cultivars. Species-diagnostic SNPs were mined from 454 amplicon sequencing of 57 gene fragments from 26 genotypes of the four basic taxa. Of the 1,053 SNPs mined from 28,507 kb sequence, 273 were found to be highly diagnostic for a single basic taxon. Species-diagnostic SNP markers (105) were used to analyse the admixture structure of varieties and rootstocks. This revealed C. maxima introgressions in most of the old and in all recent selections of mandarins, and suggested that C. reticulata × C. maxima reticulation and introgression processes were important in edible mandarin domestication. The large range of phylogenomic constitutions between C. reticulata and C. maxima revealed in mandarins, tangelos, tangors, sweet oranges, sour oranges, grapefruits, and orangelos is favourable for genetic association studies based on phylogenomic structures of the germplasm. Inferred admixture structures were in agreement with previous hypotheses regarding the origin of several secondary species and also revealed the probable origin of several acid citrus varieties. The developed species-diagnostic SNP marker set will be useful for systematic estimation of admixture structure of citrus germplasm and for diverse genetic studies.

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Changes in gene expression of Mexican lime plants in response to infection with a severe (T305) or a mild (T385) isolate of Citrus tristeza virus (CTV) were analyzed using a cDNA microarray containing 12,672 probes to 6875 different citrus genes. Statistically significant (P<0.01) expression changes of 334 genes were detected in response to infection with isolate T305, whereas infection with T385 induced no significant change. Induced genes included 145 without significant similarity with known sequences and 189 that were classified in seven functional categories. Genes related with response to stress and defense were the main category and included 28% of the genes induced. Selected transcription changes detected by microarray analysis were confirmed by quantitative real-time RT-PCR. Changes detected in the transcriptome upon infecting lime with T305 may be associated either with symptom expression, with a strain-specific defense mechanism, or with a general response to stress.

Viroids are small (246-401 nucleotides), single-stranded, circular RNA molecules that infect several crop plants and can cause diseases of economic importance. Citrus are the hosts in which the largest number of viroids have been identified. Citrus exocortis viroid (CEVd), the causal agent of citrus exocortis disease, induces considerable losses in citrus crops. Changes in the gene expression profile during the early (pre-symptomatic) and late (post-symptomatic) stages of Etrog citron infected with CEVd were investigated using a citrus cDNA microarray. MaSigPro analysis was performed and, on the basis of gene expression profiles as a function of the time after infection, the differentially expressed genes were classified into five clusters. FatiScan analysis revealed significant enrichment of functional categories for each cluster, indicating that viroid infection triggers important changes in chloroplast, cell wall, peroxidase and symporter activities.