High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

Abstract

BackgroundHigh-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT). This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera.ResultsWe have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for E. grandis. A systematic assessment of in silico SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous in silico constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species.SNP reliability was high across nine Eucalyptus species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased.ConclusionsThis study indicates that the GGGT performs well both within and across species of Eucalyptus notwithstanding its nucleotide diversity ≥2%. The development of a much larger array of informative SNPs across multiple Eucalyptus species is feasible, although strongly dependent on having a representative and sufficiently deep collection of sequences from many individuals of each target species. A higher density SNP platform will be instrumental to undertake genome-wide phylogenetic and population genomics studies and to implement molecular breeding by Genomic Selection in Eucalyptus.

Progress in Myrtaceae genetics and genomics: Eucalyptus as the pivotal genus

“…Even higher density linkage maps will in future be possible in Eucalyptus with concerted efforts of genome-wide discovery of SNP markers enabled by next-generation DNA sequencing technologies. Nevertheless, the very high frequency of SNPs observed in Eucalyptus will challenge the commonly used SNP genotyping technologies, particularly for multi-species genotyping platforms where the combined SNP diversity of several species need to be (Grattapaglia et al 2011a) accommodated (see Section "Molecular marker resources"). Ultimately, the extremely high-throughput recently achieved with next-generation DNA sequencing technologies will enable direct detection and genotyping of SNPs from short sequence tags at high coverage on reduced genomic representations of individual offspring sequenced in multiplexed barcoded pools.…”

“…Using the Sequenom mass spectrometry platform (Sexton et al 2010b) overcame this problem by generating fewer but much longer template amplicons instead of several short ones. When developing SNPs for the GGGT, stringent requirements of conservation in flanking sequences to the SNP both within and across species significantly enhanced SNP reliability and polymorphism of the assay, although such requirements reduced the number of assayable SNPs (Grattapaglia et al 2011a). This approach, although successful, showed that in silico assessment of polymorphism and interspecific transferability of the SNP requires high sequence coverage and representation to successfully provide reliable selective power for actual SNP assay development.…”

“…Recently, a dataset of >1.2 million mixed expressed sequence tags (EST) dataset including Sanger and 454 sequences from multiple Eucalyptus species was used to develop the first collection of 768 genome-wide SNPs for Eucalyptus using the Illumina GGGT (Golden Gate genotyping technology) (Grattapaglia et al 2011a). Assay success rates according to the GGGT recommended parameters were close to 95% for all five species genotyped (E. grandis, E. globulus, E. camaldulensis, E. urophylla, E. nitens).…”

Pectin Methylesterase Genes Influence Solid Wood Properties of Eucalyptus pilularis

“…This sampling of three rare alleles in PME6 was not unusual; many populations of forest trees have been found to have a high abundance of rare SNPs (with low MAF), and it is postulated that this is attributed to large population sizes and an out-crossing mating system (Eldridge et al, 1993;Novaes et al, 2008;Grattapaglia et al, 2011;T.R. Sexton, unpublished data).…”

Identification of Single Nucleotide Polymorphisms on Cattle Breeds in Indonesia Using Bovine 50k

“…Thus, the overall genotyping reliability for the total SNPs was assessed by estimating SNP counts above conventionally used threshold and average values for Call Rate, GC50 and GeneTrain scores. These measures provide some general information about quality and performance of SNPs (Illumina Inc. 2011a;Grattapaglia et al 2011). Clustering heat map and related SNP analyses were performed with GenomeStudio.…”