Association Mapping of Saccharification Yield in Sorghum Using a Mini Core Collecti

Abstract

Increasing world population and limited resources is necessarily synonymous with energy crisis. Fossilfuel has been exploited since industrial revolution and have contributed to polluting the earth to nearlethal limits. In this situation fuel from biological organisms (biofuel) is very promising in meeting theglobal energy demands and also restoring balance in the ecosystem. Biofuel is a renewable and clean source of energy. Biofuels produced from non-edible plant biomass are not yet commercially viable due to its high production cost. This study focuses on identifying genes responsible for cellulosic biofuel yield from sorghum stalks. Sorghum is a very promising energy crop as it can grow in relatively hostile conditions with low fertilizer and water input but produces high biomass nonetheless. High biomass along with highsaccharification yield (conversion of cellulosic biomass to fermentable sugars like xylose and glucose)will ensure a more cost effective biofuel. To improve saccharification yield, our lab is trying to identifycandidate genes involved in cell wall synthesis that affects saccharification yield.

Introduction

Biomass is a carbon neutral source of energy and consists of 76% of all renewable energy. Fuel produced from biomass is called biofuel and has the potential to deliver 25% of world projected energy need by 2035. The countries like Sweden , Austria , Brazil , China and USA has made progress in this technology but still biofuel is at its infancy and much research needs to be done. But critics are skeptic about its impact on land use, water and nutrient cycling, and emission of nitrous oxide.

Cellulose forms largest amount of organic matter but cellulosic biofuel production faces severe bottleneck due to recalcitrant nature of cell wall where cellulose is trapped. Cell wall especially secondary cell wall is a rich source of carbohydrate mainly composed of cellulose [a glucose polymer], xylose and lignin [phenyl propanoid polymer]. However, lignin is the main recalcitrant factor to cellulose digestion by forming crosslinks across the cell wall and shields enzyme from sugar polymers. Also lignin is found to absorb enzymes thus resulting in low saccharification yield. This can be altered by identifying and expressing genes that can alter cell wall structure or can produce more efficient cell wall degrading enzyme. The brown –midrib mutation depicts how a defect in lignin structure can improve cell wall digestibility. Rapid gene identification can be done by association mapping.

Aniruddha Acharya1*, Yi-Hong Wang1

1 Department of Biology, University of Louisiana at Lafayette

Sorghum accessions from the mini core collection [ Upadhyaya et al. 2009 ] were grown in the field, harvested, air dried for 2 weeks and cut into 20 cm pieces. They were dried again at 80°C for 2 days and milled to 2 mm with a Wiley Model 4 Laboratory Mill. The stalk powder was dried again and 0.5 g were weighed in tubes in two replicates and labeled. They were autoclaved with 5 ml of 2% sulfuric acid. The samples were immediately washed for 6 to 7 times using double distilled sterile water. Finally they were digested at 50°C shaken at 240 rpm for 24 hours. Digestion solution consisted of 5 ml citrate buffer, 50 µl Cellic CTec cellulase enzyme from Novozyme and 10 µl of 20 µg/ml tetracycline. For each sample, 100 µl of supernatant was transferred to 1.5 ml tube containing 900 µl double distilled water and mixed. 10 µl of mixed solution was used to measure glucose. Glucose content was measured using OneTouch UltraSmart® glucose meter (Figure 1; Saballos et al. 2008). Association mapping was conducted using TASSEL 3.0 (Bradbury et al. 2007; available from http://www.maizegenetics.net/) with the mixed linear model (Yu et al. 2006). 14,739 SNP markers (mostly from Wang et al. 2013) were used for the association mapping.

Materials and Methods

Sorghum stalk powder

Pretreated with 2% H2SO4

Washed and digested with CTec cellulase

Measure glucose content

Figure 1. Measuring saccharification yield in sorghum.

Result and Discussion

Glucose meter calibration

Calibration for the OneTouch UltraSmart® glucose meter using a set of D-glucose solutions in 18 MΩ water. Each standard was measured three times with similar readings (from Wang et al. 2011). Linear relationship between glucose concentration and the readings is evident.

Variation of saccharification yield

Genetic variation in saccharification yield in sorghum---the lowest and the highest yield among evaluated varieties. A. From an evaluation of the mini core varieties (Wang et al. 2011). B. From an evaluation of 426

conversion varieties (Vandenbrink et al. 2010). Name of the variety is on top of each bar.

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SNP markers strongly associated with saccharification yield in sorghumTo find genes potentially related to saccharification yield, we examined the genomic regions surrounding SNP loci associated with the trait. The gene closest to the locus on chromosome 4 codes for β-tubulin (table below). β-tubulin has been found to determine the orientation of cellulose microfibrils in plant secondary fiber cell walls and different orientation can influence the strength and flexibility of secondary plant cell walls (Spokevicius et al. 2007). The gene closest to the two SNP loci on chromosome 10 encodes a No Apical Meriatem ( NAM) transcription factor SbNAM1 ( Table below ). In Arabidopsis several closely related NAM transcription factors have been shown to be master switches of secondary wall biosynthesis ( Wang H et al. 2011 ; Zong et al. 2011). SbNAM 1 is most homologous ( 71 % identity and 74% similarity) to the maize secondary wall NAC transcription factor 1 ( ZmSWN1 ; accession number AEO53053). Overexpression of ZmSWN1 results in ectopic deposition of all three major secondary cell wall components – cellulose, xylan (hemicellulose) and lignin in the mesophyll cells which are not normally lignified ( Zhong et al 2011)..

SNP marker Association p value range SNP location (in bp) Closest gene and function Referencechr4_4361515chr4_4361522chr4_4361523chr4_4361525chr4_4361527chr4_4361534

All 7.18 10-7 except chr4_4361534 which has a p value of 2.57 10-6.

4361515 – 4361534 bp on chromosome 4

Sb04g004520 (β-tubulin)Cell wall cellulose microfibril orientation

Spokevicius et al. 2007

chr10_1801700chr10_1801720chr10_1801722chr10_1801726chr10_1801760 and chr10_1806523chr10_1806529

See Figure 2 1801700 – 1801760 bp on chromosome 10

Sb10g002120 (SbNAM1)Secondary cell wall thickening

Zhong et al. 2011

Figure 2. SNP markers associated with SbNAM1 on sorghum chromosome 10. X-axis displays the physical location and distance in bp; y-axis is the –log10(p value) as used in Figure 3---higher values indicate stronger associations. Threshold p value is 3.3910-6, the Bonferroni-corrected threshold probability at α = 0.05 as described by Murray et al. (2009) for 14,739 SNP markers. In blue are the SNP markers between 1790 kb and 1828 kb. Two annotated genes are indicated. Scale and positions of the two genes are based on information from www.phytozome.net/sorghum.

AcknowledgementsWe Dr. Susan Mopper and Andre Daugereaux for allowing the use of UL’s Center for Ecology and Environmental Technology facility. The study is supported in part by LA EPSCoR (LEQSF-EPS(2012)-PFUND-298) and the University of Louisiana at Lafayette.

Summary

We have mapped genes responsible for saccharification yield in sorghum using the mini core collection as the mapping panel and 14,739 SNP markers. The identified candidate genes have been shown to play roles in plant secondary cell wall synthesis.