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Association of genes linked to malting quality in South African barley breeding lines.

Carol Nunurai Bvindi

Masters of Science Internship report Submitted to Wageningen University and Research Centre In partial fulfilment of MSc Plant Sciences Degree Specialisation: Plant Breeding and Genetic Resources August December 2010

Department of Botany and Plant Biotechnology University of Johannesburg, South Africa

Supervisor: Dr. Eduard Venter Examiner: Prof Dr. Richard Visser

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Abstract
The malting quality of barley (Hordeum vulgare L.) is an economically important phenotype and is the key objective of barley breeding programmes. However, phenotyping for malting quality through micro-malting and micro-mashing is time consuming, labour intensive; often limited by seed availability and the procedures require expensive analyses. Hence many test procedures are done at the final stages of the breeding programme. Furthermore, it is a complex trait that represents net effects of a number of interrelated and interacting component traits each of which show quantitative inheritance. In a previous gene expression study with North American barley cultivars, at least 90 genes have shown correlation to six malting quality phenotypes. The aim of this study was to study the expression of three of these genes (-amylase, limit dextrinase, and -ketoacyl synthase) in South African barley breeding lines. -Amylase fold changes in gene expression was significantly different from Limit dextrinase, and -keto acyl synthase fold changes (P 0.05) in all the lines. Limit dextrinase and -keto acyl synthase fold changes were not significantly different from each other (P 0.05) in all lines. Sabbi-Erica showed highest fold changes in -Amylase and in Limit dextrinase gene expression. However Harrington had the highest fold change in -keto acyl synthase gene expression. Sabbi-Nemesia had the lowest fold change for Limit dextrinase and -keto acyl synthase gene expression, and Hamelin had the lowest fold change for -Amylase gene expression. Lines with high fold changes in gene expression can be profitably used as candidates for developing superior malting quality genotypes suitable for South Africa.

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Acknowledgements
My sincere gratitude goes to Dr Eduard Venter for giving the opportunity to do my internship in his laboratory and to widen my knowledge in gene expression studies. I am truly grateful for his patience and teaching abilities which I hope I didnt exhaust during my time in his laboratory. I enjoyed every bit of the learning process. I would also want to thank Sonia Greyling and Vic Nicolis for their assistance in the Lab. It was a pleasure to meet and work with such wonderful people. Last but not least I would want to thank my supervisor, Prof. Dr Richard Visser for all his help and advice especially in getting an internship position.

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Table of contents

Abstract ...................................................................................................................................................................................................... iii Acknowledgements ............................................................................................................................................................................... iv Chapter 1: Introduction........................................................................................................................................................................ 1 1. General introduction ........................................................................................................... 1 1.1 Malting quality traits ........................................................................................................ 1 1.2 Genetic components influencing malting quality............................................................. 2 1.3 RT-PCR as a method of studying gene expression .......................................................... 3 1.4 Breeding for malting quality in South Africa................................................................... 4 1.5 Research objectives .......................................................................................................... 5 Chapter 2: Materials and Methods.................................................................................................................................................. 5 2.1 Plant materials and malting conditions ............................................................................ 5 2.2 RNA extraction and cDNA synthesis............................................................................... 6 2.3 RT-qPCR analysis ............................................................................................................ 7 2.4 Data analysis .................................................................................................................... 8 Chapter 3: Results................................................................................................................................................................................... 9 3.1 Malting stages analysed in this study ............................................................................... 9 3.2 RNA extraction .................................................................................................................... 10 3.4 Optimisation of real time primers .................................................................................. 11 3.5 Gene expression fold changes at day 4 of the malting stages ........................................ 13 Chapter 4:Discussion ......................................................................................................... 15 4.1 RNA extraction .............................................................................................................. 15 4.2 Quantitative changes of -Amylase, Limit dextrinase, and -ketoacyl synthase genes in barley lines during malting. .................................................................................................. 16 4.3 Conclusion...................................................................................................................... 17 References.................................................................................................................................................................................................19

Chapter 1
1. General introduction Barley is primarily produced in South Africa for the production of malt, a substrate in brewing and fermentation of beer and distillation for the production of whiskey (USDA, 2003). During the malt production grain is steeped in water under controlled temperature and humidity to force germination that provides the necessary hydrolytic enzymes. These convert high molecular weight components in the grain like carbohydrates and storage proteins, into partially degraded biopolymers, which are subsequently utilised by enzymes in the brewing process (Potokina et al. 2004, Jones 2005). The germinated grain is then air dried in a kiln at high temperatures to halt the germination and enzyme modification processes. The entire malting process involves three main stages; steeping, germination and kilning and is well documented (Pollock 1962, Burger and La Berge 1985, Bamforth and Barclay 1993). Malting quality is of high importance to the brewing industry. It is defined by a number characteristics found both in the grain and in the malting process. Thus it is the primary objective of various barley breeding programs around the world to produce cultivars with high malting quality profiles. These profiles are defined by the brewing industry and are highly influenced by consumer preferences. Despite the industrys definition and specifications for malting quality characteristics, breeding for malting quality in barley is still a challenge. A wide range of physical and biochemical characteristics that determine the trait are influenced by environmental conditions during grain development as well as conditions during malting. Thus malting quality is a phenotype made up of many complex interrelated traits each of which have been shown to be inherited quantitavely and are of low heritability (Hayes and Jones 2000, Potokina et al. 2004, Panozzo et al. 2007). Understanding the fundamental genetic processes and the genes involved in malt production is an important factor for the development of improved malting barley varieties (Watson et al. 2005). 1.1 Malting quality traits Malting quality is dependent on a number of interrelated traits. To date, over nineteen traits have been identified to define malting quality. Among the most important traits are grain size, kernel plumpness, test weight, grain protein content, alpha () and beta () -amylase activity, diastatic power, malt extract percentage, soluble protein, ratio of soluble protein to total malt protein, malt -glucan content and wort viscosity (Fox et al. 2003, Gao et al. 2004).
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Grain size, kernel plumpness, and test weight are indicators of the uniformity of kernel size and germination which is important during the malting process (Burger and LaBerge 1985, Coventry et al. 2003.). Grain protein content is the measure of the percentage protein in the grain, usually a commercial specification for malting barley and correlates with other quality traits (Emebiri et al. 2003). - amylase breaks down (1-4) glucosidic bonds in starch, and amylase breaks down starch to maltose, a fermentable sugar. The measure of the combined effect of - amylase and -amylase activity and other carbohydrate degrading enzymes is called diastatic power (Marquez-Cedillo 2000). Malt extract percentage activity measures the amount of soluble sugars and nitrogenous compounds obtained upon mashing malt into wort. It is highly depended on the growing condition and the malting procedure (Han et al. 1997, Collins et al. 2003). Soluble protein measures the amount of protein solubilised into wort after mashing and the ratio of soluble protein to total malt protein is a measure of the extent of protein mobilization due to proteinase activity (Marquez-Cedillo 1999, Emebiri et al. 2003, Muoz- Amatrian et al. 2010). Malt -glucan content is the amount of -glucan in the wort, it depends upon the barley -glucan content and -glucanase activity during malting and it affects other traits like malt extract percentage and wort viscosity (Muoz- Amatrian et al. 2010). Wort viscosity is important for beer filtration through membranes in breweries; it is influenced by a number of parameters like -glucan content and barley cell wall polysaccharides (Fox et al. 2003). Of these many traits, the American Society of Brewing Chemists use only seven (Muoz- Amatrian et al. 2010), the Australian malting and brewing industry only considers six (Fox et al. 2003) and South African Breweries (SAB), the sole buyer of malting barley in South Africa, considers eight of these parameters of importance in defining malting quality (Potgieter and Meijering 2009). Thus the importance of these traits in defining malting quality is highly dependent on the malting conditions, brewing practises and consumer preferences. 1.2 Genetic components influencing malting quality Barley malting quality has been genetically studied for many years; many Quantitative Trait Loci (QTLs) and genes associated with malting quality have been elucidated. QTL mapping studies dissected the genomic regions associated with malting quality and at least 156 distinct malting-quality QTLs for 19 malting traits have been mapped in nine barley mapping populations (Zale 2000, Fox et al. 2003, Szucs et al. 2009). Despite this large number of QTLs, only a few of these genes have been characterised.

These are mainly genes encoding the key enzymes in malting quality pathways like starch degrading enzymes -amylase, -amylase, limit dextrinase and -glucosidase (MarquezCedillo 2000). Recent large scale gene expression technologies like cDNA arrays and serial analysis of gene expression (SAGE) have identified a number of candidate genes for malting quality (White et al. 2006, Potokina et al. 2006, Lapitan et al. 2009, Muoz- Amatrian et al. 2010). Using cDNA arrays, Potokina et al. (2004) identified 17-30 candidate genes for each of the six malting quality parameters they studied. Five out of eight of these genes displayed linkage to known QTLs for malting quality. Lapitan et al. in 2009 identified 11-102

candidate genes correlated to six malting quality phenotypes in North American barley cultivars. Using eight SAGE libraries derived from different time points during barley malting, White et al. (2006) identified 100 most abundant transcripts from each of time points. These transcripts were associated with stress and defence response, translational and hydrolytic processes occurring within the germinating seed and thus overlapped with candidate genes identified in cDNA array based studies on barley seed germination (Potokina et al. 2002). Gene expression studies provide an opportunity to identify candidate genes encoding different traits, based on the hypothesis that the observed differences at the phenotypic level are due to differences in the expression of the underlying genes. Further analysis of these candidate genes aids in production of varieties with good malting quality profiles through conventional breeding and biotechnology (Potokina et al. 2006). 1.3 RT-PCR as a method of studying gene expression Large scale gene expression technologies like cDNA arrays and SAGE are time consuming and cost intensive, hence when dealing with a smaller set of identified candidate genes Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) is the method of choice. In recent years it has been widely used in gene expression studies. The rising popularity is attributed mainly to its ability to combine quantification and detection in a single step. The technologys tremendous sensitivity, practical and conceptual simplicity, ability to quantify very small amounts, lack of post amplification processing, speed and specifity in homogeneous assays also contribute to its growing popularity (Valasek and Repa 2005, Bustin et al. 2004). In plant sciences it has been mainly applied in studying changes in gene expression in physiology, biotic and abiotic stress responses and developmental plant growth stages. It has also been used to confirm gene expression results obtained in microarray studies (Lapitan et al. 2009, Potokina et al. 2006). In this study, RT-qPCR was
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employed to study the expression of genes linked to the complex trait malting quality in South African barley breeding lines. 1.4 Breeding for malting quality in South Africa The malting barley breeding programme in South Africa is run by SAB and South African Barley Breeding Institute (SABBI) and has about twenty-seven years of existence (Potgieter and Meijering 2009). When the programme was started in 1983, it was focused on introducing new lines from other parts of the world to replace existing lines. In this light, material was collected from all over the world and evaluated for adaptability and malting quality. However, when this did not bring about the desired result, crosses were made from Australian, European and CIMMYT lines. The results of these crosses, together with the imported germplasm have been the backborne of the breeding programme. The main focus of the programme has been breeding for high fermebility, hot water extract, diastatic power, amylase activity, lower -glucans and balanced free amino nitrogen and wort viscosity (Potgieter and Meijering 2009). Techniques employed to achieve this focus have been double haploid breeding, marker assisted selection, malt fermebility indicator tests, micromalting and disease evaluations. Despite the long existence of this breeding programme, only seven cultivars have been released for malting production and currently four of these are in production (Potgieter and Meijering 2009, SABBI 2009). Development of markers for marker assisted selection with conventional methods is a challenge as the trait is complex and influenced by a number of other interrelated traits. The methods employed in assessing quality of progeny from the breeding programme by micromalting and malt fermebility indicator tests are resource intensive, time consuming and are often delayed till the later stage of the breeding programme. These challenges have limited progress and success of the breeding programme. Lack of good malting varieties together with fluctuations in weather conditions, fungal infestations, and insect damage has often resulted in the locally produced crop falling short of the malting characteristic required by the brewing industries in the past years (Potgieter and Meijering 2009, NDA 2009). As a result, barley processors in South Africa have depended on imports mostly from Canada and Australia, and to a lesser extent the European Union, to make up malting shortfalls (NDA 2009, Potgieter and Meijering 2009). Thus, there is need for more efficient and feasible techniques to identify and develop varieties with good malting characteristic in barley breeding as this is the first stage in meeting the demand of producers and processors in the South African barley industry. Identification of candidate genes through gene expression technologies and further analysis
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of these genes for their use in breeding programmes has been shown to be more feasible in breeding for complex traits like malting quality (Potokina et al. 2004). 1.5 Research objectives Based on the study by Lapitan et al. (2009), at least 90 genes were correlated to six malting quality phenotypes. These included genes involved in protein and lipid metabolism, cell wall organisation and biogenesis, stress and defence response and carbohydrate metabolism. The focus of this research was to test and associate a subset of three of these malting quality linked genes (-amylase, limit dextrinase, and -ketoacyl synthase) to the observed phenotype in South African barley breeding lines. In this study, it was reasoned that if these candidate genes for malting quality are confirmed in South African barley breeding lines, they will be useful for developing new genotypes with superior malting quality profiles suitable for South Africa.

Chapter 2

Materials and Methods


2.1 Plant materials and malting conditions Twenty-one breeding lines were obtained from SABBI, six barley grains from each line were surface sterilised in 70 % ethanol for 1 minute, washed thoroughly with sterile distilled water
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and steeped for 43 h to achieve a Relative Water Content (RWC) of 45-46 %. Steeping conditions alternated between the following: 9 h in water at 15 oC, 14 h in air at 17 oC, 14 h in water at 15 oC, 6 h in air at 15 oC. The seed where then germinated for 90 hours at 17 oC. Both steeping and germination were done at a relative humidity above 95 % and in the dark. These conditions are in accordance with the SAB micromalting conditions. Three grain samples for each line were harvested at two different stages which were 24 h after steeping (day 1) and 98 h after steeping (day 4). The harvested sample were immediately frozen in liquid nitrogen and stored at -80 oC prior to RNA isolation. Three unmalted grains from each line were used as untreated controls. 2.2 RNA extraction and cDNA synthesis Total RNA extracted according to the publication of Lapitan et al. (2009) from the seed samples did not yield pure RNA. This extraction method used the Qiagen RNeasy Plant Mini Kit with RNase Free/DNase (Qiagen) following the manufacturers instructions. The resultant carbohydrate contamination made this RNA unusable. Two other methods were tried before good quality and quantity RNA was obtained. A second method using phenol/sodium dodecyl sulphate (SDS) as published by Wilkins et al. (1996) was then tested. The third method was originally used to isolate RNA from pine trees described by Chang et al. (1993). All three these methods failed to give substantial yield and good quality RNA from the barley seeds. Modifications applied to the method of Chang et al. (1993) was combined with the RNA clean up protocol from the RNeasy plant mini kit (Qiagen, Valencia, CA, USA) was successful in extracting RNA from the barley seeds. Using this modified protocol total RNA was extracted from the 63 malted barley samples. Briefly, 100 mg of each of the samples were ground to a fine powder in liquid nitrogen and mixed completely with 500 l hot (65 oC) extraction buffer (100 mM Tris-HCl pH 8.0, 2.0 M NaCl, 25 mM EDTA, 0.5 g/L spermidine, 2.0 % CTAB and 2.0 % PVP and -mercaptoethanol added just before use). RNA was extracted two times with an equal volume of chloroform: isoamyl alcohol (24:1). The phases were separated at 10 000 rpm for 10 minutes at room temperature and the supernatant was carefully removed to a new tube. The RNA was cleaned by mixing the upper aqueous phase with 0.33 volume 3 M sodium acetate pH 5.2 and 0.5 volume 100 % ethanol, followed by incubation for 30 minutes on ice. This was then centrifuged for 25 minutes at 10 000 rpm at 4 oC to precipitate carbohydrates. The upper aqueous phase was carefully removed and mixed with 2 volumes 100 % ethanol to precipitate out total RNA. The RNA was further cleaned with the RNeasy plant mini kit (Qiagen) with DNase 1 treatment following the
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manufacturers instructions. Quantification of the total RNA was done by measuring the absorbance at 260 nm using a SmartSpecTM spectrophotometer (Bio-Rad Laboratories) and the purity was assessed by determining the ratio of the absorbance at 260 and 280 nm. To test for DNA contamination, the RNA samples were run on 1 % agarose gels and a no RT template control was included in the qPCR runs. All samples showed no DNA contamination and had 260/280 nm ratios >1.8. RNA was stored at -20 oC until cDNA synthesis. Total RNA was reverse transcribed using a commercially available cDNA synthesis kit SuperScript
TM

III Reverse Transcriptase (Invitrogen). Briefly, 11.5 L 40 g total RNA, 0.5

L 50 M oligo(dT), 1L 10 mM dNTP Mix were added to a nuclease free microcentrifuge tube and heated to 65 C for 5 minutes. The mixture was then snap cooled on ice for at least a minute followed by the addition of 4 L of 5X first strand buffer (250 mM Tris-HCL pH 8.3, 375 mM KCl, 15 mM MgCl2); 1 L 0.1 M DTT, 1 L of Rnase outTM and 1 L SuperScript
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III. The cDNA synthesis reactions were carried out in a Mycycler

TM

thermal

cycler (Bio-Rad Laboratories) by incubating at 50 C for 60 minutes, followed by the inactivation of the enzyme at 70C for 15 minutes. cDNA was stored at -20 C prior to quantitative polymerase chain reaction. 2.3 RT-qPCR analysis RT-qPCR analysis was done on day 4 cDNA based on Lapitan et al. (2009) and also to optimise our protocol. Primers for each of the 4 genes were designed by Primer 3 (Rozen and Skaletsky 1998). The primers were designed to target -amylase (F: 5-

CGGCAATGACTATGCCGTAT-3, R: 5 GCATGTCCCTCATCCTCACT-3), limit dextrinase (F: 5CGGTTTCAACACGAGGATCT-3; R: 5-ACTAGCAGCTTGGGCACTA-3), and -ketoacyl synthase: (F: 5GAGGACACAACTCGGTGGTT-3, R: 5-GCAATGGATCTTGGATCCTC-3). Quantitative PCR was performed with a 72-well Rotor-Gene 3000 (Corbett Robotics) using the 2X SensiMix DNA Kit (Quantace) according to manufacturers instructions. The total reaction volume was 10 l containing 5 l 2X Sensimix, 2 L diluted cDNA, 0.25 L of each gene specific primer (10 pmol/ L) in and 2.5 L water. The PCR reaction started by activating the hot-start DNA polymerase at 95 C for 10 minutes. This was followed by 50 cycles of target cDNA amplification with an initial denaturation at 95 C 5 s followed by annealing at 52 C for 10 s and elongation at 72 C for 5 s. Each reaction was run in duplicate both for standards and samples, as well as for negative controls.

2.4 Data analysis To generate standard curves, cDNA stock from the cultivar Kinukei 21 was used to generate a 10 fold dilution series (1:10; 1:20; 1:30; 1:40; 1:50). This dilution series was used to determine the PCR efficiency for each target gene amplification. Standard curves are used to calculate the efficiency of the qPCR assay. Efficiency is of importance when reporting RNA concentration of the target gene relative to that of the reference gene and high qPCR efficiency means that the assay is robust and precise. PCR efficiency values derived for the target gene and the reference gene together with Ct values obtained in the qPCR assay can be used to calculate fold change in gene expression. Fold change in gene expression was calculated using the Pfaffl equation below: Fold Change = (E target)CT* / (E reference)CT** Were: E is the efficiency *(CT Target gene control CT target gene sample) ** (CT Reference Gene control CT Reference Gene sample) The Pfaffl equation (Pfaffl 2001) computes the relative expression of each gene at the various stages, normalized to the expression of a reference gene. As a reference gene for normalisation the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used (Burton et al. 2004).

Chapter 3 Results
3.1 Malting stages analysed in this study The 21 lines were subjected to three malting stages as described above. It was expected that at day 1, imbibition should have taken place and the seed would have sprouted. At day 4, the shoots and roots would have elongated (Figure 1). However the 21 lines exhibited varied response at the different stages. For some (Baudin, Gairdener, Hamelin and Copeland) germination was slow, at day 4 they still had no elongated shoot only swollen seed. For some (Sabbi-Erica, S5, Marthe, Keel, Cocktail, Alexis and Xanandu) germination was not uniform, one seed would have shoots at day 4 and the rest will be still at day 1 stage. Germination rate and uniformity are some of the most important grain attributes in malting quality.

Dry seed

Day 1

Day 4

Figure 1: Barley seeds of the cultivar Harrington depicting the three different malting stages analysed on the 21 breeding lines. Dry seed is unmalted seed, day 1 is seed subjected to 24 hours of germination after steeping, and day 4 is seed subjected to 96 hours of germination after steeping.

3.2 RNA extraction The Phenol/SDS method by Wilkins et al. (1996) and the commercially available RNA extraction kit; RNeasy plant mini kit (Qiagen), gave poor quantity and quality RNA in terms of yield and A260/A280 ratio (Figure 2). No distinct 28S and 18S rRNA bands and smear in the wells on 1 % agarose gels showed that the integrity of the RNA was poor except for the modified protocol.

28S 18

Yield (ng/l): 32.85 A260/A280: 1.3

6.31 1.04

-44.95 1.22

90.96 1.92

Figure 2: RNA extracted with different protocols. Lane 1: The RNeasy mini plant kit; lane 2: Phenol/SDS protocol; lane 3: RNA isolation according to Chang et al. (1993) and lane 4: the modified protocol. Values at the bottom of each lane show the yield and quality of the RNA per 100 mg tissue.

Also the RNA from the Phenol/SDS method was contaminated with DNA (Figure 2). This poor quality RNA was unsuitable for downstream applications like RT-qPCR. The RNA extraction protocol described by Chang et al. (1993) did not give any RNA yield at all as the RNA was lost during the LiCl precipitation step. On the other hand our modified protocol produced satisfactory yield and quality RNA (Figure 2). It also gave distinct 28S and 18S rRNA bands on 1 % agarose gel. Furthermore, RT-qPCR showed that the RNA was not contaminated with DNA as the negative control (non-RT control) showed no amplified PCR product (Figure 3).

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Figure 3: RNA samples extracted from barley dry seed by the modified protocol analysed by RT-qPCR for the -amylase gene. M is the marker lane, lanes 1-4: RT-qPCR negative control (non RT control) and lane 5-7: RT- qPCR products. 3.4 Optimisation of real time primers Each primer was optimised for primer concentration and annealing temperature using a tenfold cDNA dilution series. Different primer concentrations and annealing temperatures were tried out for each primer and from each of these reactions a standard curve was generated. Optimised primers have an efficiency value close to 100 %, showing that the amount of product doubles with each real-time cycle and this is achieved if the slope is close to -3.33 is (Bustin et al. 2009). If the efficiency value was way less than 100 %, then different primer concentration and annealing temperature combinations were tried till the efficiency was close to 100 %. For each primer a standard curve was generated at optimised conditions (Figure 4). For -amylase the correlation coefficient (R2) was 0.955 and the efficiency was 82 %; for Limit Dextrinase the R2 was 0.971 and the efficiency was 88 %; for -ketoacyl synthase the R2 was 0.963 and the efficiency was 78% and for GAPDH the R2 was 0.979 and the efficiency was 87 % (Figure 4).

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Figure 4: Standard curves for A: -amylase; B: Limit Dextrinase C: -ketoacyl synthase and D: GAPDH genes. The R 2 value is the correlation coefficient and gives an indication of how close the data points fit to the standard curve. M is the slope from which the efficiency of the assay is calculated. B is the intercept points were the standard curve crosses the y axis (number of real time cycles). Efficiency is the efficiency of the qPCR assay given as the slope of a standard curve plotted as Ct values vs. log concentration.

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3.5 Gene expression fold changes at day 4 of the malting stages

Table 1: Fold change in gene expression level of -amylase, limit dextrinase, and -ketoacyl synthase at day 4 compared to the dry seeds in the 20 breeding lines based on RT-qPCR.

Fold change in gene expression Genotype Newdale SSG 564 SabbiErica SabbiNemesia S5 S9 Elandra Cocktail Metcalfe Copeland Harrington Marthe Baudin Xanadu Alexis Hamelin Kinukei 21 Keel 08-075 Gairdner amylase 162.423 1445.976 47 797.135 16979.95 162.423 17.642 0.603 111.285 1253.849 1.383 3984.656 1608.016 7.484 2177.354 25675.47 0.007 2.115 96.253 5.058 1.668 Limit Dextrinase 19.775 84.160 1260.757 0.217 376.460 51.363 44.820 8.815 5.557 4.607 49.119 1.949 1.422 17.995 65.542 2.538 71.719 337.593 16.721 2.360 keto acyl synthase 0.712 10.565 0.059 0.001 14.607 11.316 84.364 5.334 1.502 0.863 403.792 11.948 0.026 60.501 0.433 0.023 0.670 2.610 2.621 0.117

Changes in gene expression level of the three genes (-Amylase, Limit dextrinase, and ketoacyl synthase) at day 4 compared to the dry unmalted seed for each line was analysed. For all three genes there was a positive fold change in all the breeding lines indicating upregulation of the genes at day 4 compared to the dry seed. In all the lines fold changes in genes expression was varied, with the -Amylase fold changes in gene expression
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significantly different from Limit dextrinase, and -keto acyl synthase fold changes (P 0.05) in all the lines. Limit dextrinase and -keto acyl synthase fold changes were not significantly different from each other (P 0.05) in all lines. -Amylase fold change in gene expression ranged from 0.007 to 47 797.135, Limit dextrinase fold change in gene expression ranged from 0.217 to 1260.757 and -keto acyl synthase fold changes ranged from 0.001 to 403.92 (Table 2). -keto acyl synthase fold changes were the lowest compared to the fold changes in expression of the two other genes. Sabbi-Erica showed highest fold changes in -Amylase and in Limit dextrinase gene expression. However Harrington had the highest fold change in -keto acyl synthase gene expression. Sabbi-Nemesia had the lowest fold change for Limit dextrinase and -keto acyl synthase gene expression, and Hamelin had the lowest fold change for -Amylase gene expression (Table 1).

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Chapter 4 Discussion
4.1 RNA extraction The major problem encountered when extracting RNA from barley seeds is the high level of polysaccharides in the grain. Polysaccharides tend to form complexes with nucleic acids, coprecipitating with them thus rendering unavailable in solution. The RNeasy plant mini kit (Qiagen,) has Extraction Buffers (EB) that contains guanidine salts (guanidine thiocyanate and guanidine hydrochloride). These compounds results in starch swelling, making the extraction mixture thick glue like and ultimately results in solidification of the extract (Li et al. 2005). This results in co-precipitation of RNA with starch and subsequently contamination of the extracted RNA (Singh et al. 2003). This also explains the poor quantity and yield of RNA extracted using this kit. The Phenol/SDS method also gave unsatisfactory results due to the fact that SDS results in starch swelling as described above and is thus unsuitable for starch rich tissues such as seeds (Kurakake et al. 2004). To exacerbate the problem, phenol can be easily oxidised to form covalently bonded quinones which binds irreversibly to nucleic acids (Chang et al. 1993, Meng and Fieldman 2009). The RNA isolation of Chang et al. (1993) overcame the problem of starch co-precipitating with the RNA as it included polyvinylpyrrolidone (PVP) and -mercaptoethanol in the extraction buffer. These compounds act as reducing agents and thus separate starch and proteins from nucleic acids (Chang et al. 1993, Birtic and Kranner 2006). The method also extracted the RNA with chloroform instead of phenol, getting rid of polyphenols and polysaccharides thus making the RNA available in solution. Furthermore the EB had a high pH which is suitable to inhibit RNase activity. However this protocol resulted in significant RNA loss during the LiCl precipitation step. Highly concentrated salts, like LiCl, have been shown to significantly reduce yield and quality of RNA particularly from barley seeds (Singh et al. 2003). Thus in our modified protocol, the extracted RNA was first cleaned by preferentially precipitating the polysaccharides with volume of ethanol. The resultant RNA was then precipitated with 2 volumes ethanol and cleaned with RNeasy spin columns instead of precipitating with LiCl. This was successful in obtaining better yield and quality RNA from the barley seeds. Ethanol has been shown to be effective in concentrating and de-salting nucleic acids preparations in aqueous solution. Addition of ethanol forces nucleic acid to precipitate out of solution (Zeugin and Hartley 1985), the precipitate can easily bind to the

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RNeasy spin column membrane, washed with the kits wash buffers and then eluted in RNase free water. 4.2 Quantitative changes of -Amylase, Limit dextrinase, and -ketoacyl synthase genes in barley lines during malting. In comparison to other studies, fold changes in several of the lines was exceptionally high. For example Harrington fold change for -Amylase gene was 3984.656 in this study, and in a study by Lapitan et al. (2009) was 6.00. Considering the fact that almost similar condition was used between the two experiments, our fold change was off. This indicates some uncertainties in our data. It also indicates that there is need for further optimisation of the qPCR assays as the qPCR efficiencies used in the data analysis were quite low less that 90 %. Low efficiencies according to according to Bustin et al. (2009) increases the chances of false positives. -Amylase gene expression fold change at day 4 compared to the dry seed was shown to be high in most of the lines. This is not a new phenomenon as -Amylase plays an active role in seed germination and is synthesized de novo in the cells of the aleurone layer in response to gibberellins secreted by the embryo upon germination (Ho 1979, Hayter and Riggs 1973, Potokina et al. 2002). However, -Amylase activity also constitutes the diastatic power measurement which is a measure of the malt capacity to hydrolyse starch into fermentable sugars. Thus it is an important quality factor in malting barley and can be used to effectively select for varieties with good malting profiles (George-Kraemer et al. 2001, Lapitan et al. 2009). The lines with high fold changes in -Amylase gene expression can be used profitably as candidates for good malting quality profiles varieties. This is in accordance with Sekiguchi et al. (1984) who concluded that selection for high -Amylase activity can be effective in breeding for better malting quality varieties. Variation in -Amylase fold change between the lines is consistent with other studies (Sekiguchi et al. 1984, Arends et al. 1995, GeorgeKraemer et al. 2001). In these studies they attributed the variation to genotype and the environment. However in our case, as the environment was constant for all the lines, it can be reasoned that genotype was the main source of variation between the lines. The Limit dextrinase gene encode for -dextrin endo-1,6--glucosidase, an enzyme that debranches amylopectin and dextrins formed during germination or malting and complete the depolymerisation of starch to glucose (Li et al. 1995). In all the lines there was a positive fold change in the expression of the Limit dextrinase gene. This is profitable for the breeding programme as high expression levels mean high activity of encoded enzyme. High enzyme
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activity will increase hydrolysis of dextrins to fermentable sugars, improving wort fermentability and ultimately an increase in alcohol yield (Longstaff and Bryce 1993, Li et al. 1995, Burton et al. 1999). Also, Limit dextrinase is the only enzyme that is able to degrade 1,6 linkages in amylopectin (Li et al. 1995), and considering the fact that barley grain contains 75 % branched amylopectin (Kasha et al. 1993), Limit dextrinase is an important factor in malting quality noteworthy to select for in breeding for malting quality. Noteworthy is that the line (Sabbi-Erica) with the highest fold change in Limit dextrinase expression is the one of the lines with the best malting profile currently used in production of malting barley in South Africa (SABBI 2009). This depicts the importance of Limit dextrinase in malting quality. -keto acyl synthase fold changes in gene expression were the lowest amongst the cultivars compared to the fold changes of the other genes. Contradicting results in whether -keto acyl synthase is differentially expressed during malting have been reported. Lapitan et al. (2009) showed that it was not differentially expressed during the malting stages of the four cultivars they studied using microarrays. However when the same experiment was repeated using RTqPCR, the gene expression was significantly among the four cultivars. This gene plays a role in the chain elongation step in fatty acid synthesis leading to palmitoyl-ACP and stearoyl ACP. However its role in malting quality has not yet been elucidated (Lapitan et al. 2009). In our case, -keto acyl synthase may play an unknown role in malting quality as its fold change in expression in the cultivar Harrington is significantly higher compared to the other lines. Harrington has one of the best malting profiles, and is used as background in many malting quality breeding lines. 4.3 Conclusion In conclusion, this research has devised a modified protocol for RNA extraction from barley seeds. This protocol, we believe can be used effectively for RNA extraction from other materials with high starch content. Furthermore we identified breeding lines with high expression levels of two of the most important genes in malting quality (-Amylase and Limit dextrinase). The obtained results may already indicate lines that can be used as candidates for breeding for cultivars with superior malting quality profiles. However the results of this study are preliminary as there is need for further studies, such as association studies of the expressed genes with the observed phenotypes, to fully realise their use in breeding malting quality. It is recommended that confirmation of any expression data should be verified with additional association studies to identifying more candidate lines to be used in the breeding
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programme. The very high expression levels in several of the cultivars indicates that not too much should be concluded from these data as there are discrepancies in the expression levels obtained in this study in comparison to other studies. Also studying the expression of the other identified genes linked to malting quality in these lines will further help in identifying lines with superior malting quality.

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