Divergence of barley and oat varieties according to their content of β-glucan

Barley and oat have been promoted for years as good sources of dietary fiber, especially β-glucan. Studies have shown a positive effect of barley and oat β-glucan on human health and as a result, there has been an increasing demand for β-glucans in the past decade. Thus, the variability of the β-glucan content was investigated in the grain of 10 barley and 10 oat varieties. The β-glucan contents were determined by the ICC Standard Method No 168. The content of β-glucan in the analyzed barley varieties were within the range 3.52–7.81 % and in analyzed oat varieties, the content of β-glucan were within the range of 3.15–7.28 %. Among analyzed barley varieties, Novosadski 314 contained the highest content of β-glucan (7.81 %), while Tomba had the highest content of β-glucan (7.28%) in the analyzed oat varieties. Based on the results, it could be concluded that there is genetic diversity of oat and barley varieties with respect to their β-glucan content. This fact enables varieties with a high a nutritional capacity to be selected.


INTRODUCTION
Barley and oat have been promoted for years as good sources of dietary fiber, especially β-glucan. 1,2β-1,3/1,4-glucans are polysaccharides present in the bran of some grains, such as oat and barley but to a much lesser degree in rye and wheat. 3In seeds of barley and oat, the highest concentrations of β-glucans are found in the endosperm and the subaleurone layer of the seed. 4,5Numerous studies are increasingly showing a positive effect of β-glucan from barley and oat on human health.7][18] Furthermore, β-glucans may represent an important component of food in the modification of metabolic irregularities and the prevention of metabolic syndrome. 19,20Based on in vitro experiments, and animal and human clinical studies, there is considerable evidence concerning the importance of β-glucan in the treatment and prevention of allergic diseases, which opens new perspectives in the use of this group of natural substances. 21he physiological effects of β-glucans are mainly attributed to their physicochemical structure, such as molecular weight, viscosity, solubility and interaction with the gastrointestinal tract. 22,235][26] Among other things, β-glucans, which exist as indigestible polysaccharides in barley and oat, have been shown not only to have positive effects on health, but also the potential to be a possible new source of prebiotic. 27,28nowing these beneficial effects of β-glucan, it is important to determine the availability and the best potential sources of β-glucan and possibly to increase them, in order to promote their wider application and accessibility in everyday life.
Considering that the content of β-glucan in oat and barley could be increased by breeding and using appropriate agro-technology (effect of fertilizationapplication of nitrogen (N) affects positively by increasing the content of β-glucan) [29][30][31] whereby the effect of genotype is dominant and has been confirmed, 29,32 the goals of this research were to define the content of β-glucan in genetically divergent cultivars of 10 oat and 10 barley varieties, comparison of the β-glucan content in different oat and barley varieties, and identification of the varieties with higher contents of β-glucan as a modest contribution to the breeding of oat and barley.

Plant material
The plant material used for the field trials consisted of 10 varieties of spring barley (Hordeum sativum J. ssp.distichum L.) and 10 varieties of spring oat (Avena sativa L.).These genotypes originated from the Gene Bank collection of Field and Vegetable Crops in Novi Sad.Two rows of spring barley varieties were used for investigation, i.e., Novosadski 296, Novosadski 297, Novosadski 300, Novosadski 301, Novosadski 306, Novosadski 310, Novosadski 312, Novosadski 314, Novosadski 316 and Novosadski 318, and 10 varieties of spring oat, i.e., Flaemingsterne, Vok, Mozart, Juha, Hannes, Alden, Aslak, Wasa, Tomba and Kaempe gul.The barley and oat cultivars used for analysis were employed in increase of the production of cultivar seeds, because of their high genetic potential of yield, quality and adaptability.Nowadays, these cultivars are used in cross hybridization and represent the core gene source in breeding programs for the creation of new enhanced cultivars.

Field experiment
The experiment design in 3 replications was a basic plot with 5 rows of 1 m length with an inter-row spacing of 0.2 m and in row distance of 0.05 m between the seeds.Sowing was performed by hand.The trial was realized in the location Kragujevac (44°00′51′′ N, 20°54′42′′ E), during two growing seasons -2011 and 2012.In the first year of the experiment, phenotypes were estimated and biotypes for each variety removed.In the second year of the experiment, the seed material was used for the determination of β-glucans.Mineral fertilizers (NPK 15:15:15, MAP -monoammonium phosphate) were applied before seeding according to the recommendations, based on the chemical properties of the soil and the available content of P, K and N reserves.The following fertilizers were applied for nutrition: urea (46 % N), KAN (27 % N) and AN (34 % N).The crop was protected adequately against pests and weeds by the appropriate use of pesticides and herbicides, with their efficacy being monitored.For analysis of the grain, full maturity stage of 30 plants (10 plants per replication) was used.

Soil characteristics
The growing of barley and oat genotypes was realized on an experimental field on the soil type pseudogley having poor physical properties, an acid pH (pH H 2 O 4.1) and the following content: humus 2.34 %, readily available phosphorus 7.8 mg 100 g -1 soil and potassium 14.2 mg 100 g -1 soil.

Climatic conditions during growing seasons
The values of temperature and precipitation in two years of experiment were different in relation to the average values of the previous ten years (Table I).During the period January/June, the average values of the temperature was 9.28 °C in the first and 9.38 °C in the second year of the experiment.In both years of the experiment, the average temperature was lower than in the long-term period (10.08 °C).The precipitation was more favorable in the year 2011, with sums of 327.9 mm, than in 2012 (378.9 mm).In the second year during the grain filling stage (April-May months), the amount of precipitation (174.6 mm) was higher than in the first year (120 mm) and in the second year, the amount of precipitation was similar to the sum of the long term precipitation (373.7 mm).

Seed sample
After harvesting all the genetically divergent barley and oat varieties, 5 g of seeds were used for grinding and for analysis of their content of β-glucans.

Laboratory analysis
The β-glucan contents were determined by the ICC Standard Method No 168, which was adapted to measure the content of β-glucan in barley, oat and their products.Particles smaller than 500 µm were used in the experiment.

Processing of the results
Results for content of β-glucan of analyzed barley and oat varieties were obtained by spectrophotometrically reading the absorbance at λ=510 µm for each of the three test tubes (Table II).The final content of β-glucan was obtained by applying the following formula for grains of barley and oat: β-glucan (mass %) = ∆A×F×94 (or 64)×0.10w×162/180= ∆A×F/W×8.46(or 5.76) (1)   where ∆A is the absorbance after β-glucosidase treatment (reaction); F -a factor for the conversion of absorbance values to µg of glucose: Absorbance of 100 μg of -glucose and 94 is a volume correction factor (0.1 ml out of 9.4 ml was analyzed for cereal samples); 64 is a volume correction factor (0.1 ml out of 6.4 ml was analyzed for cooked, toasted of extruded cereal products); 0.10w is a factor to express the β-glucan content as a percentage of dry flour weight; W is the calculated dry weight of the sample analyzed in mg; 162/180 is a factor to convert from free D-glucose, as determined, to anhydro-D-glucose, as occurs in β-glucan 33 and w is the grain weight.
Based on the β-glucans contents, all the analyzed barley varieties were compared with each other and a similarity dendrogram with Euclidean distances was made (Fig. 1).An analysis of the dendrogram showed two clusters (groups) with mutually similar varieties (Fig. 1).In the first cluster were the two barley varieties Novosadski 296 and Novosadski 316, with a distance of about 41 % (the degree of similarity was 59 %).The second cluster consisted of a larger number of varieties and pairs showing a high percentage of similarity (Novosadski 297, β-GLUCAN FROM BARLEY AND OAT 383  Novosadski 301, Novosadski 306, Novosadski 310, Novosadski 300, Novosadski 314 and Novosadski 318).In the second cluster, the greatest similarity, 99 %, was observed for the varieties Novosadski 306 and Novosadski 310.Another pair consisted of the varieties Novosadski 297 and Novosadski 301 with a similarity of 97 %.With the previous pair of varieties of the second cluster, they form a small group with a distance of 8 % (degree of similarity of those pairs is 92 %).
The most similar to this group according to values of β-glucans was Novosadski 300 with distance in the range of 22 %.The second cluster with a distance of 22 % also contains a pair of varieties, Novosadski 314 and Novosadski 318, which are 97.5 % similar.The degree of similarity of the first and second cluster is 51 % (49 % distance).The variety Novosadski 312 stands out because of the greatest distance (82.5 %) compared to all other barley varieties.

The content of β-glucans in oat
The content of β-glucan in the analyzed oat varieties ranged from 3.15 to 7.28 % (Table II).The highest content of β-glucan was found in the variety Tomba, with a concentration of 7.28 %.A lower content of 5.6 % was found in the variety Vok.The oat varieties in which the content of β-glucan ranged from 4 % to 5% were Flaemingsterne 4.79 %, Juha 4.71 %, Wasa 4.71 %, Kaempe gul 4.37 %, Hannes 4.25 % and Mozart 4.23 %.Among the remaining analyzed oat varieties that have a β-glucan concentration of less than 4 % are Alden (3.38 %) and Aslak (3.15 %), in which the lowest concentration of β-glucan was observed.The average value of the β-glucans contents in the analyzed oat varieties was 4.65 %.Based on the β-glucans contents, all the analyzed oat varieties were compared with each other and a similarity dendrogram with Euclidean distances was made (Fig. 2).An analysis of the dendrogram shows two clusters (groups) with mutually similar varieties (Fig. 2).The first cluster consisted of two small groups of varieties, which together show a high percentage of similarity.The first group includes the varieties Flaemingsterne, Juha and Wasa, with the highest percentage of similarity (99 %) within the first cluster.Within the second group, there is a pair of varieties Mozart and Hannes with a degree of similarity of 98 %.The variety Kaempe gul joins this pair with a distance of 8 %.The degree of similarity between the first and second group is 80 % (the distance between mentioned groups is 20 %).The second cluster includes the two oat varieties Alden and Aslak with a mutual distance in the range of 14 %.The degree of similarity of the first and second cluster is 50 %.The variety Vok stands out from the two clusters by a distance in the range of 52 %, while the most notable variety is Tomba due to the greatest distance (97 %) compared to all other varieties of oat.
Many studies have shown that the grains of barley and oat are characterized by higher concentrations of β-glucans compared to other grains, whereby barley has a higher content of β-glucan with respect to oat.The average concentration of β-glucan in barley is approximately in the range of 3 to 7 %, whereas the concentration of β-glucan in oat is approximately in the range of 2 to 6 %. [34][35][36] Havrlentova and Kraic noted a large variation in the content of β-glucan in barley and oat varieties in all genotypes and indicated that barley and oat may be convenient sources of β-glucan. 34ased of the biochemical analysis of the β-glucan content in the investigated oat and barley varieties, it could be concluded that the content of β-glucan was higher in the barley varieties than in the oat varieties, which could be observed through the average value of the β-glucan contents (6.6 % of the dry weight of the grain in the analyzed barley varieties, but only 4.65 % of the dry weight of the grain of the analyzed oat varieties).In the tested barley varieties, the β-glucan content ranged from 3.52 to 7.81 % of the dry grain weight, while the β-glucan content of analyzed oat varieties ranged from 3.15 to 7.28 % of the dry grain weight.The highest contents of β-glucan in the analyzed barley varieties were found in Novosadski 314 (7.81 %) and Novosadski 318 (7.8 %), while the highest content of β-glucan in analyzed oat varieties was observed in Tomba, with a concentration of 7.28 %.Numerous studies suggest the beneficial effects of β-glucans on human health as immunostimulants, 3,7,8 they can act both anti-mutagenic and anti-carcinogenic, 10,11,37 they can be used for lowering cholesterol levels and improvement of serum lipid status, [38][39][40] they can have a positive effect on glycemic and insulin response, 17,18 and among others, β-glucans can represent a new source of prebiotics. 27,28As a result of their positive effects on health, there has been an increasing demand for β-glucans in the last decade.For this reason, it is necessary to find the most affordable sources of β-glucans to enable their application and ensure their availability.
The variability of the β-glucan content is controlled by both genetic and environmental factors and their interaction, which acts during the period of endosperm development. 36,41,42mong the environmental factors, warm and dry weather during grain-filling results in higher levels of β-glucan, while high precipitation or irrigation are det-rimental. 42,43][31] Considering that genotype had the greatest influence on variability than all the other factors and their interactions, the dominant effect of which has been confirmed in numerous studies, 29,32,41 the selection of the proper genotype is the most crucial for desired amount of β-glucan in grain. 32[39] Based on the obtained results, it could be concluded that genetic diversity influences the content of β-glucan in oat and barley varieties, giving the possibility of choosing varieties for the selection of lines of high nutritional capacity required to improve nutrition and to use as a source for obtaining useful preparations in the pharmaceutical industry for further implementation.
The varieties that could be recommend for hybridization and breeding are barley varieties Novosadski 314 and Novosadski 318 and oat varieties Tomba and Vok, as varieties with the highest content of β-glucans.

Fig. 1 .
Fig. 1.Dendogram of the genetic distances between the clusters of barley varieties for their content of β-glucan (the dendogram was obtained with Statistica for Windows, version 10).

Fig. 2 .
Fig. 2. Dendogram of the genetic distances between clusters of oat varieties for their content of β-glucan (the dendogram was obtained with Statistica for Windows, version 10).

TABLE I .
Monthly and mean temperatures and monthly and cumulative precipitation; Xmaverage values of temperature and precipitation

TABLE II .
The content of β-glucan in oat and barley varieties (mass %)