Approaches to variant discovery for conifer transcriptome sequencing

Abstract

There is a wide diversity of bioinformatic tools available for the assembly of next generation sequence and subsequence variant calling to identify genetic markers at scale. Integration of genomics tools such as genomic selection, association studies, pedigree analysis and analysis of genetic diversity, into operational breeding is a goal for New Zealand’s most widely planted exotic tree species, Pinus radiata. In the absence of full reference genomes for large megagenomes such as in conifers, RNA sequencing in a range of genotypes and tissue types, offers a rich source of genetic markers for downstream application. We compared nine different assembler and variant calling software combinations in a single transcriptomic library and found that Single Nucleotide Polymorphism (SNPs) discovery could vary by as much as an order of magnitude (8,061 SNPs up to 86,815 SNPs). The assembler with the best realignment of the packages trialled, Trinity, in combination with several variant callers was then applied to a much larger multi-genotype, multi-tissue transcriptome and identified 683,135 in silico SNPs across a predicted 449,951 exons when mapped to the Pinus taeda ver 1.01e reference.

Effect of trait’s expression level on single-step genomic evaluation of resistance to Dothistroma needle blight

“…Genomic data were generated through exome capture -genotyping-by-sequencing approach [21] using genomic resources based on resequencing of transcriptome extracted from compression wood xylem, spring xylem, summer xylem, summer phloem, spring buds, autumn buds, healthy needles, needles infected by Phytophtora pluvialis, seedling phloem and seedling xylem [66]. Captured markers were removed if heterozygosity shown in megagametophyte tissues was higher than 5%, average read depth less than 10, multiallelic status, singletons and additionally each datapoint was classified as missing if ratio between reference and alternative allele was lower than 0.1 and number of read was less that 10 [67].…”

A high-density exome capture genotype-by-sequencing panel for forestry breeding in Pinus radiata

“…A first set of 30 samples was used to obtain some preliminary performance metrics for the pilot probe panel, and consisted of: a) genotypes used to generate the exome templates [26] for capture probe design; b) ‘trios’, consisting of both parents and a single offspring to identify probes with non-Mendelian inheritance; c) six ‘duo’ parent and megagametophyte combinations to identify probes capturing paralogous regions; d) technical replicates of two unrelated genotypes to identify probes that did not yield repeatable results; and e) an additional replicate, prepared using an alternative DNA extraction protocol.…”

“…In Pinus flexilis E. James, transcriptomics-based resequencing reported 3.7 SNPs per kilobase [24]; in P. taeda L., Lu et al [25] reported 11.5 SNPs per kilobase. Within P. radiata , SNP discovery via transcriptomic resequencing across 8 genotypes delivered 328,981 SNPs across 449,951 putative exons [26]. Even though more SNP markers are required than microsatellites for an equivalent level of discriminatory power, there is continual development of multiple platforms for capturing SNP genotypes, allowing for the cost-effective development of both high and low throughput assays [27].…”

“…To date, a number of conifer programmes have developed transcriptome-based SNP resources for breeding applications, including Douglas-fir ( Pseudotsuga menziesi Mirb.) [44], maritime pine ( Pinus pinaster Aiton) [45] and radiata pine ( Pinus radiata D.Don) [26]. There are a number of genotyping platforms suitable for assaying genome-wide markers including both fixed SNP arrays (Illumina [46], Affymetrix [47]) and genotype-by-sequencing (GBS) based approaches [48, 49].…”

“…There are a number of genotyping platforms suitable for assaying genome-wide markers including both fixed SNP arrays (Illumina [46], Affymetrix [47]) and genotype-by-sequencing (GBS) based approaches [48, 49]. We have chosen an exome capture GBS method that takes advantage of a previously developed transcriptome-based SNP resource in this species [26]. This method, which sequences portions of genes from across the genome was developed in P. taeda [50], and successfully developed for other large conifers, as well such as Norway spruce ( Picea abies ) [51] and Douglas-fir ( P. menziesii ) [52].…”