Use of Targeted Amplicon Sequencing in Peanut to Generate Allele Information on Allotetraploid Sub-Genomes

Abstract

The use of molecular markers in plant breeding has become a routine practice, but the cost per accession can be a hindrance to the routine use of Quantitative Trait Loci (QTL) identification in breeding programs. In this study, we demonstrate the use of targeted re-sequencing as a proof of concept of a cost-effective approach to retrieve highly informative allele information, as well as develop a bioinformatics strategy to capture the genome-specific information of a polyploid species. SNPs were identified from alignment of raw transcriptome reads (2 × 50 bp) to a synthetic tetraploid genome using BWA followed by a GATK pipeline. Regions containing high polymorphic SNPs in both A genome and B genomes were selected as targets for the resequencing study. Targets were amplified using multiplex PCR followed by sequencing on an Illumina HiSeq. Eighty-one percent of the SNP calls in diploids and 68% of the SNP calls in tetraploids were confirmed. These results were also confirmed by KASP validation. Based on this study, we find that targeted resequencing technologies have potential for obtaining maximum allele information in allopolyploids at reduced cost.

Keywords: allopolyploid; heterozygous SNP calls; targeted resequencing; tetraploids.

Figure 1

GATK pipeline for SNP identification and target selection (for the OLin method, the SNP identification pipeline would be the same expect for the alignment with the OLin transcriptome sequence instead of the synthetic tetraploid genome reference).

Figure 2

MiSeq Sequencing Run Distribution of reads by Peanut Accession on MiSeq (250X2) (total reads: 30,153,796; PF Reads (Reads that have passed Illumina quality filter): 24,430,536) (each index corresponds to a different plant accession, given in Supplemental Table S5). Red bars indicate the accessions that failed to amplify satisfactorily.

Figure 3

Example of validation of SNP calls derived from MiSeq using KASP markers.

Figure 4

Target sequence with multiple SNPs and indels. (a)—An example showing homoelogous (top) and homoeologous (bottom) SNPs in tetraploids. Yellow rectangles at left denote tetraploids. Red, blue, and green rectangles (top) denote A, B, and K genome diploids, and red and blue ovals denote A and B genomes in tetraploids. Allele calls are shown above the figure. At bottom are homologous SNPs differentiating tetraploids; SNPs are denoted above the figure as alleles 1 or 2 (orange and purple ovals at left), and 3 or 4 (pink or cyan ovals at right). The order of sequences is the same in the top and bottom figures. (b) An example of target sequence in IGV viewer showing multiple SNPs (There are two SNPs showing up in all accessions (blue and orange), which may be referred to as anchor SNPs). The target SNP is highlighted by a red circle.