Genome-wide SNP discovery in walnut with an AGSNP pipeline updated for SNP discovery in allogamous organisms

Abstract

BackgroundA genome-wide set of single nucleotide polymorphisms (SNPs) is a valuable resource in genetic research and breeding and is usually developed by re-sequencing a genome. If a genome sequence is not available, an alternative strategy must be used. We previously reported the development of a pipeline (AGSNP) for genome-wide SNP discovery in coding sequences and other single-copy DNA without a complete genome sequence in self-pollinating (autogamous) plants. Here we updated this pipeline for SNP discovery in outcrossing (allogamous) species and demonstrated its efficacy in SNP discovery in walnut (Juglans regia L.).ResultsThe first step in the original implementation of the AGSNP pipeline was the construction of a reference sequence and the identification of single-copy sequences in it. To identify single-copy sequences, multiple genome equivalents of short SOLiD reads of another individual were mapped to shallow genome coverage of long Sanger or Roche 454 reads making up the reference sequence. The relative depth of SOLiD reads was used to filter out repeated sequences from single-copy sequences in the reference sequence. The second step was a search for SNPs between SOLiD reads and the reference sequence. Polymorphism within the mapped SOLiD reads would have precluded SNP discovery; hence both individuals had to be homozygous. The AGSNP pipeline was updated here for using SOLiD or other type of short reads of a heterozygous individual for these two principal steps. A total of 32.6X walnut genome equivalents of SOLiD reads of vegetatively propagated walnut scion cultivar ‘Chandler’ were mapped to 48,661 ‘Chandler’ bacterial artificial chromosome (BAC) end sequences (BESs) produced by Sanger sequencing during the construction of a walnut physical map. A total of 22,799 putative SNPs were initially identified. A total of 6,000 Infinium II type SNPs evenly distributed along the walnut physical map were selected for the construction of an Infinium BeadChip, which was used to genotype a walnut mapping population having ‘Chandler’ as one of the parents. Genotyping results were used to adjust the filtering parameters of the updated AGSNP pipeline. With the adjusted filtering criteria, 69.6% of SNPs discovered with the updated pipeline were real and could be mapped on the walnut genetic map. A total of 13,439 SNPs were discovered by BES re-sequencing. BESs harboring SNPs were in 677 FPC contigs covering 98% of the physical map of the walnut genome.ConclusionThe updated AGSNP pipeline is a versatile SNP discovery tool for a high-throughput, genome-wide SNP discovery in both autogamous and allogamous species. With this pipeline, a large set of SNPs were identified in a single walnut cultivar.

Novel Roles for the Polyphenol Oxidase Enzyme in Secondary Metabolism and the Regulation of Cell Death in Walnut

“…Because the walnut genome has not yet been sequenced, reads from RNA-seq analysis were aligned to a previously assembled and annotated walnut transcriptome database containing 85,045 contigs (You et al, 2012). Substantial expression ($20 aligning reads) was detected for 53,994 of these contigs in our data set.…”

“…Trimmed reads were aligned to a walnut transcriptome assembly consisting of 85,045 contigs using BurrowsWheeler Aligner version 0.6.1 (Li and Durbin, 2009). The walnut transcriptome was previously assembled from approximately 1 billion 85-nucleotide RNA-seq reads derived from RNA isolated from 20 different walnut tissue samples (You et al, 2012). The BLAST2GO program (Conesa et al, 2005) was utilized for putative functional annotation of the walnut sequences.…”

Genome wide SNP discovery in flax through next generation sequencing of reduced representation libraries

“…The discovery of single nucleotide polymorphic (SNP) markers combined with next generation sequencing (NGS) permits the identification of thousands of markers from entire genomes which can be used for linkage map construction, genetic diversity analyses, marker-trait association and marker-assisted selection [21]. SNPs have been discovered by high throughput sequencing in humans [22], Drosophila melanogaster [23], wheat [24], eggplant [25], rice [26-28], Arabidopsis thaliana [29,30], barley [31-33], walnut [34], lupin [35], globe artichoke [36], rapeseed [37], perennial ryegrass [38] and maize [39] to name but a few. SNP discovery through genome sequencing is readily accomplished in simpler genomes like rice and Arabidopsis [28,40] but the task remains challenging for a number of economically important crops [41,42].…”

“…The objective of the current study was to discover and validate SNPs in flax using a combined NGS of RRLs and GBS strategy with the updated annotation based genome-wide SNP discovery pipeline (AGSNP) [34,61]. The resource promises to have several downstream applications including the exploitation of flax genetic diversity through the understanding of important phenotypic traits.…”

Analysis of the Chickpea genome using next generation sequencing data

“…Some software are available with sequencing packages such as CASAVA (consensus assessment of sequence and variation). Some commercial software such as NextGENe, CLC Genomics workbench and free-ware program such as SNPdetector (Zhang et al, 2005), ACCUSA (Frohler and Dieterich, 2010), AGSNP (You et al, 2012), PolyScan (Chen et al, 2007), samtools (http://samtools.sourceforge.net/), GATK (Zhu et al, 2015) and SGSautoSNP (Lorenc et al, 2012). AutoSNP (Batley et al, 2003b;Duran et al, 2009a) was extended as AutoSNPdb (Duran et al, 2009c;Savage et al, 2005) and can now integrate both Sanger and Roche 454 data for SNP discovery.…”