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Identification of genes involved in (1,3;1,4)-β-glucan synthesis
Miriam Schreiber
MonoGram 2015 Conference, Rothamsted
(1,3;1,4)-β-glucan
Acts as a dietary fibre and promotes health benefits
Negative influence on brewing and distilling processes
Grain (1,3;1,4)- β-glucan content in grasses
Burton RA and Fincher GB (2012) Front Plant Sci
Species
(1,3:1,4)- B glucan
content (%w/w)
DP3:DP4
ratio
Oats 6 - 8 1.5 - 2.3 : 1
Barley 4 - 10 2.6 : 1.0
Wheat 1 3.2 : 1.0
Brachypodium 40 5.9 : 1.0
Rice <0.06 1.0 : 1.4
Maize Trace 2.5 : 1.0
Sorghum 0.07 - 0.2 2.8 : 1.0
Houston et al. (2014) BMC Genomics
%
Cellulose synthase-like F
Schreiber et al. (2014) PLOS One
HvCslF6 is essential
Taketa et al. (2012) J Exp Bot Transforming Nicotiana benthamiana with a
functional HvCslF6 resulted in detection of
β-glucan
HvCslF6 has been shown to be essential for β-glucan synthesis
GWAS of grain (1,3;1,4)-β-glucan content
Houston et al. (2014) BMC Genomics 1H 2H 3H 4H 5H 6H 7H
a) Spring 2- row eigenanalysis
b) Winter 2- row eigenanalysis
-lo
g10
(P)
-lo
g10
(P)
1H 2H 3H 4H 5H 6H 7H
Aim
Identifying the pathway upstream of HvCslF6
eQTL study combined with co-expression
Stem β-glucan content
Introducing the dataset
144 spring barley cultivars
grown in a polytunnel at the James Hutton Dundee side, Spring 2011
Harvesting of the 2nd internode
RNA extraction and processing by Jason Kam
Sequencing at TGAC on Illumina HiSeq
Micha Bayer
Mapping and SNP evalutation
Helena Oakey
Adjusting expression for batch differences and normalization to DESeq
Excluding two cultivars (library issues and expression differences)
Reduced dataset to 142 cultivars
Claire Halpin’s Lab
Genotype data
new RNA SNP-marker
9K iSelect + SNPs from the existing BOPA
(Comadran et al. (2013) Nature Genetics)
69,591 Marker (SNP marker and iSelect marker)
Dataset
Minor allele frequency > 5%
Missing data < 20%
Imputed missing values, using R impute
25,186 marker
Dataset
Phenotype data
Expression for 21,294 genes
Excluded genes with not enough variation
16,746 genes
eQTL
Expression QTL
Expression of HvCslF6 as trait (Phenotype)
cis- and trans-eQTLs
cis-eQTLs: ~5 Mbp around your gene of interest, highest effects
trans-eQTLs: distributed across the genome
Using a Mixed Linear Model (MLM) in GAPIT (genome association and
prediction integrated tool)
Lipka et al. (2012) Bioinformatics
eQTL analysis of HvCslF6
no cis-eQTL
Co-expression analysis
Calculating the Pearson
Correlation Coefficient
GO annotation
Carbohydrate metabolic process
Located at Golgi or plasma membrane
Most seem to be involved in primary cell wall biosynthesis
eQTL on co-expressed genes
Formin-like
Glycosyltransferase 43
Summary & Outlook
Summary
Identified a region of interest on chromosome 4H
Toolset to check for genes involved in (1,3;1,4)-β-glucan synthesis
Outlook
Developing of a CRISPR-Cas9 system for gene editing of some of the identified candidates
Acknowledgment
University of Dundee The James Hutton Institute
ARC CoE Plant Cell Walls
Jason Kam Kelly Houston George Dimitroff
Helena Oakey Micha Bayer Alan Little
Abdellah Barakate Runxuan Zhang Rachel Burton
Claire Halpin Jennifer Stephens Geoff Fincher
David Marshall
Robbie Waugh
Funding: RESAS, Strategic partnership in food and drink