Towards a molecular taxonomic key of the Aurantioideae subfamily using chloroplastic SNP diagnostic markers of the main clades genotyped by competitive allele-specific PCR.

BACKGROUND: Chloroplast DNA is a primary source of molecular variations for phylogenetic analysis of photosynthetic eukaryotes. However, the sequencing and analysis of multiple chloroplastic regions is difficult to apply to large collections or large samples of natural populations. The objective of our work was to demonstrate that a molecular taxonomic key based on easy, scalable and low-cost genotyping method should be developed from a set of Single Nucleotide Polymorphisms (SNPs) diagnostic of well-established clades. It was applied to the Aurantioideae subfamily, the largest group of the Rutaceae family that includes the cultivated citrus species. RESULTS: The publicly available nucleotide sequences of eight plastid genomic regions were compared for 79 accessions of the Aurantioideae subfamily to search for SNPs revealing taxonomic differentiation at the inter-tribe, inter-subtribe, inter-genus and interspecific levels. Diagnostic SNPs (DSNPs) were found for 46 of the 54 clade levels analysed. Forty DSNPs were selected to develop KASPar markers and their taxonomic value was tested by genotyping 108 accessions of the Aurantioideae subfamily. Twenty-seven markers diagnostic of 24 clades were validated and they displayed a very high rate of transferability in the Aurantioideae subfamily (only 1.2 % of missing data on average). The UPGMA from the validated markers produced a cladistic organisation that was highly coherent with the previous phylogenetic analysis based on the sequence data of the eight plasmid regions. In particular, the monophyletic origin of the "true citrus" genera plus Oxanthera was validated. However, some clarification remains necessary regarding the organisation of the other wild species of the Citreae tribe. CONCLUSIONS: We validated the concept that with well-established clades, DSNPs can be selected and efficiently transformed into competitive allele-specific PCR markers (KASPar method) allowing cost-effective highly efficient cladistic analysis in large collections at subfamily level. The robustness of this genotyping method is an additional decisive advantage for network collaborative research. The availability of WGS data for the main "true citrus" species should soon make it possible to develop a set of DSNP markers allowing very fine resolution of this very important horticultural group.

Genotyping by sequencing reveals the interspecific C. maxima / C. reticulata admixture along the genomes of modern citrus varieties of mandarins, tangors, tangelos, orangelos and grapefruits.

The mandarin horticultural group is an important component of world citrus production for the fresh fruit market. This group formerly classified as C. reticulata is highly polymorphic and recent molecular studies have suggested that numerous cultivated mandarins were introgressed by C. maxima (the pummelos). C. maxima and C. reticulata are also the ancestors of sweet and sour oranges, grapefruit, and therefore of all the "small citrus" modern varieties (mandarins, tangors, tangelos) derived from sexual hybridization between these horticultural groups. Recently, NGS technologies have greatly modified how plant evolution and genomic structure are analyzed, moving from phylogenetics to phylogenomics. The objective of this work was to develop a workflow for phylogenomic inference from Genotyping By Sequencing (GBS) data and to analyze the interspecific admixture along the nine citrus chromosomes for horticultural groups and recent varieties resulting from the combination of the C. reticulata and C. maxima gene pools. A GBS library was established from 55 citrus varieties, using the ApekI restriction enzyme and selective PCR to improve the read depth. Diagnostic polymorphisms (DPs) of C. reticulata/C. maxima differentiation were identified and used to decipher the phylogenomic structure of the 55 varieties. The GBS approach was powerful and revealed 30,289 SNPs and 8,794 Indels with 12.6% of missing data. 11,133 DPs were selected covering the nine chromosomes with a higher density in genic regions. GBS combined with the detection of DPs was powerful for deciphering the "phylogenomic karyotypes" of cultivars derived from admixture of the two ancestral species after a limited number of interspecific recombinations. All the mandarins, mandarin hybrids, tangelos and tangors analyzed displayed introgression of C. maxima in different parts of the genome. C. reticulata/C. maxima admixture should be a major component of the high phenotypic variability of this germplasm opening up the way for association studies based on phylogenomics.