Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 ›...

15
Identification of genes involved in (1,3;1,4)-β-glucan synthesis Miriam Schreiber MonoGram 2015 Conference, Rothamsted

Transcript of Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 ›...

Page 1: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

Identification of genes involved in (1,3;1,4)-β-glucan synthesis

Miriam Schreiber

MonoGram 2015 Conference, Rothamsted

Page 2: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

(1,3;1,4)-β-glucan

Acts as a dietary fibre and promotes health benefits

Negative influence on brewing and distilling processes

Page 3: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

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

%

Page 4: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

Cellulose synthase-like F

Schreiber et al. (2014) PLOS One

Page 5: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

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

Page 6: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

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

Page 7: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

Aim

Identifying the pathway upstream of HvCslF6

eQTL study combined with co-expression

Stem β-glucan content

Page 8: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

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

Page 9: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

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

Page 10: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

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

Page 11: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

eQTL analysis of HvCslF6

no cis-eQTL

Page 12: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

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

Page 13: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

eQTL on co-expressed genes

Formin-like

Glycosyltransferase 43

Page 14: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

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

Page 15: Identification of genes involved in (1,3;1,4)-β-glucan ... › docs › MN2015 › O5.4_Miriam.pdf · Wheat 1 3.2 : 1.0 Brachypodium 40 5.9 : 1.0 Rice

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