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Development of SNP Markers for Genes of the Phenylpropanoid Pathway and Their Association to Kernel and Malting Traits in Barley

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Development of SNP Markers for Genes of the Phenylpropanoid Pathway and Their Association to Kernel and Malting Traits in Barley

Manuela Peukert et al. BMC Genet.

Abstract

Background: Flavonoids are an important class of secondary compounds in angiosperms. Next to certain biological functions in plants, they play a role in the brewing process and have an effect on taste, color and aroma of beer. The aim of this study was to reveal the haplotype diversity of candidate genes involved in the phenylpropanoid biosynthesis pathway in cultivated barley varieties (Hordeum vulgare L.) and to determine associations to kernel and malting quality parameters.

Results: Five genes encoding phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H) and dihydroflavonol reductase (DFR) of the phenylpropanoid biosynthesis pathway were partially resequenced in 16 diverse barley reference genotypes. Their localization in the barley genome, their genetic structure, and their genetic variation e.g. single nucleotide polymorphism (SNP) and Insertion/Deletion (InDel) patterns were revealed. In total, 130 SNPs and seven InDels were detected. Of these, 21 polymorphisms were converted into high-throughput pyrosequencing markers. The resulting SNP and haplotype patterns were used to calculate associations with kernel and malting quality parameters.

Conclusions: SNP patterns were found to be highly variable for the investigated genes. The developed high-throughput markers are applicable for assessing the genetic variability and for the determination of haplotype patterns in a set of barley accessions. The candidate genes PAL, C4H and F3H were shown to be associated to several malting properties like glassiness (PAL), viscosity (C4H) or to final attenuation (F3H).

Figures

Figure 1
Figure 1
Biosynthetic pathway of flavonoids with the investigated genes labeled in red. Abbreviations of the enzymes are: PAL – phenylalanine ammonia-lyase; C4H – cinnamate 4-hydroxylase; 4CL – 4-coumaroyl-CoA ligase; CHS – chalcone synthase; CHI – chalcone isomerase; F3H – flavanone 3-hydroxylase; DFR – dihydroflavonol reductase.
Figure 2
Figure 2
Structures of the sequenced phenylalanine ammonia-lyase (PAL) gene fragments PAL_1 and PAL_2. Comparison of both fragments revealed differing SNP patterns indicating to belong to different gene copies of the PAL gene family. Exons are depicted as light grey boxes. Blue – CAPS marker available, green – high-throughput SNP marker developed, red arrows – shared SNP-positions of both investigated gene fragments.
Figure 3
Figure 3
CAPS assays for SNP 1 (a) and SNP 4 (b) of the C4H_1 gene fragment. The restriction pattern is derived by using the enzymes AccI for SNP1 and TaqI for SNP4, and is shown for 16 reference genotypes. M = 1 kb marker.
Figure 4
Figure 4
Genetic structure of the flavanone 3-hydroxylase (F3H) gene fragments F3H_1 and GM022. Light grey boxes represent exons and white boxes represent introns. The thin line indicates the non-sequenced region. ID – Insertion/Deletion, blue – CAPS marker, green – high-throughput SNP marker.
Figure 5
Figure 5
Restriction pattern observed by the CAPS marker DFR_ ID1 developed for the two bp InDel in the dihydoflavonol reductase (DFR) encoding gene, shown for 16 reference genotypes. M = 1 kb marker.
Figure 6
Figure 6
Genetic mapping of the candidate genes from the Phenylpropanoid pathway: phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H) and dihydroflavonol reductase (DFR). All gene fragments were mapped using the Steptoe x Morex mapping population, except DFR which was mapped on chromosome 3H* using the Morex x Barke population (kindly provided by Nils Stein, IPK Gatersleben).
Figure 7
Figure 7
Level of kernel raw protein assigned to a) F3H haplotypes 1 and 3 and b) to the allelic distribution of SNP38 detected in the F3H encoding gene. ┴ = minimum values, ┬ = maximum values, boxes are 0.25 to 0.75 quartiles including ─ = median.

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