Multi-Species SNP Discovery from 240 Eucalyptus Resequenced Genomes: Development and Validation of a Eucalyptus 60,000 SNP Chip, the "EucHIP60k.BR"

Dramatic progress and dropping prices of sequencing now allows SNP discovery based on resequencing whole genomes of large numbers of individuals. Differently from genome complexity reduction-based SNP discovery, this approach mitigates SNP ascertainment bias and maximizes homogeneous genome coverage. We used this approach to develop a multi-species high density Infinium chip for Eucalyptus. Genomes of 240 unrelated trees were sequenced (~4X) covering samples of the 11 most widely planted species across three subgenera (E. grandis, E. urophylla, E. globulus, E. pellita, E. pilularis, E. camaldulensis, E. benthamii, E. nitens, E. dunnii, E. cloeziana, E. tereticornis) and the related Corymbia citriodora. Sequence data (1.2 Tbp) was subject to a customized pipeline for short-read alignment, variant discovery and recalibration of SNP metrics using >160k high quality SNPs derived from deep RAD-sequencing data. SNP selection also involved aspects such as polymorphism within and across species, flanking SNPs to the target SNP, genome-wide distribution and a GATK score based on SNP sequence context. From a total of 46,997,586 raw SNP variants, a final list of 70,482 SNPs was sent to manufacture and 60,441 provided high quality genotypes in more than 15,000 samples of >25 Eucalyptus species genotyped to date.  SNP density (1 SNP/~10kbp) closely follows gene density, providing a superb tool for GWAS and Genomic Selection applications. The EucHIP60K.br is the highest density SNP chip for any tree species to date.  A comparative analysis of the in silico predicted and in vitro converted SNPs provided a roadmap for similar multi-species SNP platform developments in other plant genera.