Targeted amplicon sequencing + next-generation sequencing\u2013based bulked segregant analysis identified genetic loci associated with preharvest sprouting tolerance in common buckwheat (Fagopyrum esculentum)

Ryoma Takeshima,Katsuhiro Matsui,Yasuo Yasui,Eri Ogiso-Tanaka

doi:10.1186/s12870-020-02790-w

Abstract

BackgroundCommon 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.ResultsBy 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.ConclusionsWe 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.

Highlights

Common buckwheat (2n = 2x = 16) is an outcrossing pseudocereal whose seeds contain abundant nutrients and potential antioxidants
We efficiently developed genetic maps using a method combined with AmpliSeq technology and next-generation sequencing (NGS)-bulked-segregant analysis (BSA), and detected quantitative trait locus (QTL) associated with preharvest sprouting tolerance in common buckwheat
Distribution frequencies of preharvest sprouting (PHS) in three populations We investigated the distribution frequencies of PHS tolerance in parental lines and five F2 populations developed by self-pollination of each F1 plant derived from crosses between the highly PHS-tolerant lines ‘Kyukei 29’ (KY29), ‘Kyukei 28’ (KY28), and NF1 and the PHS-susceptible SC line Kyukei SC7’ (KSC7) (Fig. 2; Additional files 1 and 2: Table S1, S2)

Summary

Introduction

Buckwheat seeds contain health-promoting compounds with antioxidative, antihypertensive, and anti-obesity properties [1, 2], in addition to high levels of starch and high-quality protein with a well balanced amino acid profile [3, 4]. These beneficial compounds are strongly influenced by external effects; in particular, germination on the plant, called preharvest sprouting (PHS), severely degrades seed quality. Improving PHS tolerance of buckwheat is a major breeding target worldwide

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