Targeted amplicon sequencing + next-generation sequencing–based bulked segregant analysis identified genetic loci associated with preharvest sprouting tolerance in common buckwheat (Fagopyrum esculentum)

Abstract

Background Common buckwheat (2n = 2x = 16) is an outcrossing pseudocereal whose seeds contain abundant nutrients and potential antioxidants. As these beneficial compounds are damaged by preharvest sprouting (PHS) and PHS is likely to increase with global warming, it is important to find efficient ways to develop new PHS-tolerant lines. However, genetic loci and selection markers associated with PHS in buckwheat have not been reported. Results By next-generation sequencing (NGS) of whole-genome of parental lines, we developed a genome-wide set of 300 markers. By NGS- based bulked segregant analysis (NGS-BSA), we developed 100 markers linked to PHS tolerance. To confirm the effectiveness of marker development from NGS-BSA data, we developed 100 markers linked to the self-compatibility (SC) trait from previous NGS-BSA data. Using these markers, we developed genetic maps with AmpliSeq technology, which can quickly detect polymorphisms by amplicon-based multiplex targeted NGS, and performed quantitative trait locus (QTL) analysis for PHS tolerance in combination with NGS-BSA. QTL analysis detected two major and two minor QTLs for PHS tolerance in a segregating population developed from a cross between the PHS-tolerant ‘Kyukei 29’ and the self-compatible susceptible ‘Kyukei SC7’. We found different major and minor QTLs in other segregating populations developed from the PHS-tolerant lines ‘Kyukei 28’ and ‘NARO-FE-1’. Candidate markers linked to PHS developed by NGS-BSA were located near these QTL regions. We also investigated the effectiveness of markers linked to these QTLs for selection of PHS-tolerant lines among other segregating populations. Conclusions We efficiently developed genetic maps using a method combined with AmpliSeq technology and NGS-BSA, and detected QTLs associated with preharvest sprouting tolerance in common buckwheat. This is the first report to identify QTLs for PHS tolerance in buckwheat. Our marker development system will accelerate genetic research and breeding in common buckwheat.

Agrigenomic Diversity Unleashed: Current Single Nucleotide Polymorphism Genotyping Methods for the Agricultural Sciences

“…For this reason, the following sections focus more specifically on the described example cases and how each group selected SNPs, designed primer pools, ran multiplex PCR, and performed sequencing. Variations of these methodologies have been applied in agricultural sciences many times [58][59][60][61][62][63][64]. Below are four different pipelines developed for targeted sequencing genotyping.…”

“…In some situations, it may be more advantageous for some groups to outsource the steps of library preparation and sequencing. A small amount of analysis and bioinformatics would be required in-lab to interpret results [58][59][60][61][62][63][64]. Additional example use-cases of sequencing-based genotyping include the following.…”

Genetic basis of maturity time is independent from that of flowering time and contributes to ecotype differentiation in common buckwheat (Fagopyrum esculentum Moench)

“…We previously performed next-generation sequencing (NGS)-based bulked segregant analysis (NGS-BSA) with the Ion AmpliSeq targeted sequencing technology (Thermo Fisher Scientific, Waltham, MA, USA) to rapidly construct a genetic map and conduct QTL analysis [24]. In that study, we used two types of custom AmpliSeq marker sets: a target trait-linked marker set developed from NGS-BSA data and a genome-wide marker set.…”

“…3). Both our linkage map included a larger number of usable markers (or loci) and had a smaller average marker distance between loci than previous maps, such as those constructed with 269 cleaved-amplified polymorphic sequences and insertion/deletion (indel) markers (752.5 cM) and an F 2 population [18], AmpliSeq markers (550.1 cM) and an F 2 population [24], and 346 loci (773.8 cM) or 410 loci (800.4 cM) provided by microarray analysis of an F 1 pseudo-test cross [38]. GRAS-Di-CDG performed an additional estimation step for the co-dominant marker genotype based on a mixture of distributions of amplicon read counts.…”

“…The genetic analysis of common buckwheat is still challenging because of its heteromorphic self-incompatibility (SI) with two types of floral architecture: thrum (short style) and pin (long style) [23]. To allow genetic analysis in F 2 populations, we previously used the self-compatible common buckwheat line 'Kyukei SC7' (KSC7), developed by introducing the self-compatibility (SC) allele from a wild relative, F. homotropicum, and identified a genetic region related to preharvest sprouting tolerance [24]. We also developed a genome-wide marker set based on the Single Nucleotide Polymorphism (SNP) information of parental lines.…”