Assessment of phenotypic and storage protein diversity in exotic barley cultivated in District Dir ( Pakistan )

University of Malakand, Chakddara, 18800, Khyber Pakhtunkhwa, Pakistan. Tel.: +92-346-934-29-56. E-mail: muradsahil77@gmail.com, mhussainpk8@yahoo.com Ali, M., Nisar, M., Khan, W., Naz, T., Zaman, S. U., Hussain, M. (2019). Assessment of phenotypic and storage protein diversity in exotic barley cultivated in District Dir (Pakistan). Regulatory Mechanisms in Biosystems, 10(4), 400–405. doi:10.15421/021960


Introduction
Barley is economically significant, mostly for the production of malt and food. The value of this crop has increased due to its consumption as a functional food with a range of beneficial effects on health (Hua et al., 2015). Application of barley such as malt in brewing, animal feed, human consumption and the distilling industry make it a globally important crop. The long process of breeding has given rise to an increase in diversity of barley varieties (Ivandic et al., 2003). Cultivators choose the best variety of barley according to specific climate and its applications such as the distilling industry, human consumption, animal feeding and malt preparation (Ferreira et al., 2016). Genetic diversity incorporates new feasible sources in plant breeding to continue increasing the grain yield, plant height and quality. There have been many studies involving testing of barley diversity through many parameters in which genetic diversity in both wild and cultivated barley has been reported (Baik & Ullrich, 2008;Yahiaoui et al., 2008;Ledovskoy et al., 2010;Khodayari, 2012;Nandha & Singh, 2014).
Barley (Hordeium vulgare L.) belongs to the genus Hordeium of the grass family; the plant height is from 20 to 60 cm. The barley plant's stem is erect and hollow, with cylindrical internodes, each node bearing leaves; two types of root system have been found, seminal and adventitious. Leaves produced on the stem are alternate and about 5-15 mm wide. The structure of the leaves is composed of ligules, sheath blade and auricle. Barley differs from other cereal crops due to its smooth auricle and ligule that envelop the stem and have anthocyanin pigment (Gomez-Macpherson, 2001). Barley has oval shape grain, which narrows at each end. Protective exterior husks are present surrounding the grain while huskless varieties of barley are also found. However the huskless varieties are not appropriate for agriculture (Derbew et al., 2013). Barley (H. vulgare L.) is one of the major crops of the world; barley is a rich source of carbohydrates (80%) and energy. The ancient Romans also used barley for energy. Gladiators were fed on barley so they were called "hordeari" from hordeium.
Researchers have found that due to consistency of non-starch polysaccharides in barley, also known as glucans, it is able to lower the blood serum cholesterol level in humans (Weber et al., 1991). The barley grain comprises approximately 10-17% protein, 2-3% lipids, 65-68% starch (main carbohydrate), 1.5-2.5% minerals and about 4-9% B glucans while soluble dietary fibers present 3-20% in total of 11-34% dietary fibers. B glucans are the major non-starch polysaccharides in barley grain (Tsen, 1985;Marathée & Gomez-MacPherson, 2001). On the basis of production, after wheat, maize and rice, barley is considered the fourth most important cereal crop of the world and was ranked in the list of top ten crop plants in the world. Barley grows well in the cool season, the optimum temperature for its growth is about 15-30 ºC. About 59% and 14% of total barley is used as feed and in industries, respectively (Toivonen et al., 2013). Similarly, it has been used to resolve the taxonomic and evolutionary problems of several plants (Vapa & Radovic, 1998;Ali et al., 2018) due to high polymorphism. Recently, both wild and cultivated barley have been characterized on the basis of seed storage protein by using SDS-PAGE. The aim of this assay is adaptation and release of new cultivars superior in their yield contributing traits and having the ability of adapting to climate.

Materials and methods
For the present research assay, 198 exotic accessions of H. vulgare L. were selected from the Gene Bank of PGRI (Plant Genetic Resources Institute) NARC (National Agriculture Research Center) Islamabad, Pa-kistan. The catalogue sorted information shows that these selected accessions are from other agro-climate countries i.e. Japan, China, Iran, Cyprus, Syria, and the USA, which represents a wide range of ecological zones from dry mountains to irrigated plains.
Morphological characterization. During the present research work, a total of 26 morphological traits were observed in order to assess genetic diversity. The morphological traits were divided into qualitative and quantitative traits. A total of 17 qualitative traits include growth habit, stem pigmentation, auricle pigmentation, photoperiod sensitivity, kernel covering, spike density, aleurone colour, grain colour, lemma colour, lemma awn, lemma awn barbs, awn colour, glume and awn. glume colour, lemma type, length of rachilla hairs, row number. 9 quantitative traits include day to germination, days to flowering, days to maturity, plant height, biomass per plant, seed per spike, spike length, 100 seed weight and harvest index.
Biochemical characterization. For protein extraction five healthy seeds from each genotype were taken for polyacrylamide gel electrophoresis through SDS-PAGE. The grain was ground to fine powder, 0.3 g of powder was taken from each sample. We added 700 µL protein extraction buffer and vertex to homogenize. The powder was centrifuged at 15000 rpm for 40 minutes at 40 ºC. 12.5% polyacrylamide gel containing separation gel (0.4% SDS, 3M Tris-HCL and PH 9) and stacking gel (0.4% SDS, 0.4M Tris-HCL, PH 3.4) were used.0.125% SDS, 129 M Glycine, 0.025 M Tris and run on 80 V for 5 hrs. The gel was then kept in de-staining solution for 8 hours, until the blue background disappeared and bands become visible. The data were analyzed on SPSS 16.0 and Statistica 10.

Result
A total of 26 agro-morphological characters were studied, which are further divided into qualitative and quantitative traits. The 9 quantitative traits include days to germination, days to flowering and maturity, 100 seed weight, spike length, seed per spike, plant height, plant biomass, and harvest index. The 17 qualitative traits include growth habit, stem pigment, auricle pigmentation, photoperiod sensitivity, spike density, lemma awn, lemma type, lemma colour, glume and awn, glume colour, lemma awn barbs, awn colour, kernel covering, grain colour, length of rachilla hairs, alurone colour and row number.
Qualitative traits. In the present research work 17 qualitative traits were noted. Growth habit, in total 17.04% landraces showed erect growth and 16.52% had intermediate growth type while prostrate growth type was observed in 0.00% landraces. Three types of pigment were observed on the stem i.e. green, basal purple and more purple. In the total of 198 alien genotypes, 16.18% showed basal purple pigmentation, 14.99% showed green and 2.21% showed more purple type of stem pigmentation. 28.61% showed green pigmentation, 1.70% had pale purple, 1.70% showed purple and 1.02% showed dark purple pigmentation in the auricle. Photoperiod sensitivity of plants was grouped into four categories i.e. low, very low, intermediate and very high. 28.45% of plants showed intermediate sensitivity, 2.56% showed very high, 2.56% low, while very low was shown by 0.00% plants. On the basis of frequency and percentage distribution, spike density was divided into three categories i.e. weak, intermediate and high. 15.33% showed intermediate spike density, 14.31% had weak while 3.75% landraces showed high density. In total 198 accessions of barley lemma awn were also observed. In these 20.61% had awns, 12.61% awnlets while 0.00% were awnless. Three types of lemma awn barbs were observed, smooth, intermediate and rough. A total of 33.39% had rough, 0.17% had intermediate while 0.00% had smooth lemma awn barbs.
On the basis of frequency and percentage distribution glume and awn were divided into four categories i.e. length of glume and awn shorter than kernel, length of glume and awn longer than kernel, glume plus awn shorter than kernel, glume plus awn twice as long as kernel. 16.35% had glume length and awn longer than kernel, 4.26% glume plus awn shorter than kernel, 1.36% length of glume and awn shorter than kernel and 0.85% glume plus awn twice as long as kernel. In case of glume colour there was no variation found. All the landraces showed white colour of glume. In the 198 lines, three types of lemma were observed. 27.26% had lemma teeth, 2.90% showed lemma hair and 0.34% had no lemma teeth. Four types of awn colour were observed. On the basis of frequency and percentage distribution 21.12% showed amber/white colour, 6.64% showed yellow colour, 4.77% showed reddish colour 1.02% showed brown while black colour was found in 0.00%.In total of 198 accessions of barley 19.93% showed long rachilla hairs while 13.62% had short rachilla hairs. Kernel covering was divided into three categories i.e. naked grain, semi covered grain and covered grain. 2.39% had naked grain and 31.81% had covered grain while semi covered grain was observed in 0.00%. 31.86% showed white grain colour, 0.85% showed purple colour, 0.34% had red colour of grain and 0.51% had black grain colour. No variation was found in aleurone colour. All were white in colour.
Quantitative traits. Days to germination of barley was divided into three categories (low intermediate and high), the minimum number of days to germination was 130 while the maximum was 155 with mean value 140.2, sample variance 57.8 and standard deviation 7.60. In case of days to flowering, the samples were placed into three categories (low, intermediate and high) the maximum range for days to flowering was 22 while minimum range was 9 with sample variance 5.9, standard deviation 2.43 and mean value 9.7. 100 seed weight also divided into three categories ( Correlation analysis of quantitative traits. Correlation was calculated by using SPSS software for 9 morphological characters of barley accession. In total 36 coefficients of value were observed in which 19 were highly positively correlated while 17 was negatively correlated. The highly positive correlation was observed for days to germination with days to flowering 0.0576, seeds per spike 0.0537 and harvest index 0.386. Days to flowering show positive correlation with spike length 0.104 and plant height 0.045 while 100 seed weight shows positive correlation with plant height 0.225, spike length 0.059, biomass per plant 0.096, seed per spike 0.1066 and harvest index 0.0304. Spike per plant shows positive correlation with spike length 0.001, plant height 0.108 and harvest index 0.208 while spike length shows positive correlation with plant height 0.073, days to maturity 0.049 and harvest index 0.010. Positive correlation of plant height was observed with biomass per plant 0.209 whereas biomass per plant shows positive correlation with days to maturity 0.011. The positive correlation of days to maturity was observed with harvest index 0.001. Highly negative correlation was shown by days to germination with 100 seed weight -0.020, spike length -0.023, plant height -0.080, biomass per plant -0.190, days to maturity -0.090 (Table 2).
Cluster analysis. The genetic tree based on protein binary data matrix divides barley into multiple clusters (Fig. 2) on the basis of variation present in their protein profile. It was observed that the dendrogram delineated accessions into four linkages (L-1, L-2, L-3 and L-4). All the four linkages further consist of 12 clusters. Cluster-1 of Linkage-1 consists of 17 accessions out of 198, in which 2 genotypes come from the USA, 13 genotypes from Japan and 2 from Syria. Cluster-2 of Linkage-1 comprises 11 genotypes in which 8 come from Japan while 3 from Syria. Cluster-3 consists of 17 genotypes out of which 14 come from Japan while 3 come from Syria. Cluster-4 of the same linkage contains total 6 genotypes including 5 from Japan and 1 from Syria. Cluster-5 of Linkage-2 comprises 23 genotypes out of the total of 198 lines, in which 1 comes from the USA, 1 from China, 3 from Iran, 3 from Syria and 15 from Japan. Cluster-6 consists of 14 lines, out of which 11 come from Japan, 2 from the USA and 1 from Syria. Cluster-7 of the same linkage had only 17 lines, including 14 from Japan and 3 from Syria.Cluster-8 of Linkage-3 consists of 25 genotypes, out of which 12 come from Japan, 6 from Syria, 6 from Iran and 1 from Cyprus. Cluster-9 contains a total of 21 genotypes, in which 10 come from Japan, 9 from Syria, 1 from Iran and 1 from Cyprus. Cluster-10 comprises 14 genotypes, in which 8 lines come from Japan, 5 from Syria and 1 from Cyprus. Cluster-11 of the same linkage contain 13 lines including 8 genotypes from Japan, 4 from Syria and 1 from Cyprus. Cluster-12 of Linkage-4 comprises 17 genotypes out of 198, lines in which 15 belong to Japan and 2 are from Syria (Fig. 2).

Discussion
In the present research work, 198 genotypes of barley were assessed to identify genetic variability among genotypes of barley with the help of morphological traits including both qualitative and quantitative traits. A total of 26 morphological traits were studied, out of which there were 9 quantitative traits including days to germination, days to flowering, plant height, spike length, seeds per spike, 100 seed weight, biomass per plant days to maturity and harvest index. The 17 qualitative traits included growth habit, stem pigment, auricle pigmentation, photoperiod sensitivity, spike density, lemma awn, lemma awn barbs, glume and awn, glume colour, lemma type, awn colour, length of rachilla hairs, kernel covering, lemma colour, grain colour, alurone colour and row number.
On the basis of frequency distribution, significant variation was found in qualitative traits except for aleurone colour and glume colour which showed no variation. All the landraces showed white colour of glume and aleurone. In the present research work, photoperiod and spike density were also observed. Two types of growth habit was observed, erect and intermediate. In cases of pigmentation, three types of pigment were observed on the stem; green, basal purple and more purple while on the auricle purple, pale purple and dark purple pigmentation was observed. Three type of lemma was observed in which some had lemma teeth, some showed lemma hair, while some had no lemma teeth. Lemma awn barbs showed three types, smooth intermediate and rough. In the case of awn and lemma colours, four colours were observed including amber/white, yellow, reddish and brown awn while lemma showed normal, purple, red and black colour. According to the present study, the barley accessions had long and short rachilla hairs. With kernel covering, covered grain and naked covered grains were observed. In the present study, white, purple, red and black grain were observed. The results of the present study were strongly supported by the work of Zeeshan et al. (2012), who worked on 151 genotypes of barley for agro-morphological evaluation. A total of 11 qualitative traits were recorded including row type, spike density, glume awn, glume colour, grain colour, awn colour, awn barbs, kernel covering, lemma colour and rachilla hairs.
During the present work a remarkable variability was observed among the different genotypes of barley landraces. A significant variation was reported in spike length (4-914 cm) and 100 seed weight (29.2-90.0 g) as was also found in the work of (Zeeshan et al., 2012), who reported spike length 11.87-45.17 cm and 100 seed weight range 1.43-6.76 g. In the present observations, plant height ranges from 3-55 cm while days to maturity range from 165-186. Similarly (Sipahi et al., 2010) also stated a wide range of variation for plant height 44.9-94.1 cm, days to maturity ranging 92-131 and grain yield. During the present research work on barley landraces seeds per spike ranged from 18-840, biomass per plant 4-484.4, days to germination 130-155, days to flowering ranged from 9-22 and harvest index from 9.75-2100.00. Ebrahim et al. (2015) also reported variability in spike length, number of seeds per spike, plant height, number of spikelets per spike, and weight of seed per spike, days to maturity, thousand seed weight, biomass yield, and grain yield of barley landraces.
Seed storage protein profile is used to solve the evolutionary and taxonomic difficulties of many crops (Ladinzishy & Hymowitz, 1979). In the present study SDS analysis was done for 198 genotypes, in which total 26 bands were observed and all were polymorphic, as also found in the work of (Sipahi et al., 2010), who observed 15 bands in his experiment on 34 genotypes of barley. Another botanist (Baloch et al., 2014) worked on 90 genotypes of barley and observed 20 bands. (Mzid et al., 2016) also reported 37 bands in his research on barley landraces. In the present study, a high level of variation was observed in B26 (0.98%) and B25 (0.89%). This variation expresses the genetic diversity present in seed storage protein. This provides an advantage to plant breeders for future breeding activities. Through PCORD software a scatter plot and dendrogram tree was constructed which showed significant variation among the genotypes. PCA divided landraces into multiple groups on the basis of similarity and dissimilarity. In the present study, the dendrogram tree separated landraces into four linkages i.e. L-1, L-2, L-3 and L-4, which is further divided into 12 clusters. Linkage-1 consists of 4 clusters i.e. cluster-1, cluster-2, cluster-3 and cluster-4. Linkage-2 consists of 3 cluster i.e. cluster-5, cluster-6 and cluster-7. Linkage-3 consists of cluster-8, cluster-9, cluster-10 and cluster-11. Linkage-4 consists of cluster-12 only. PCA is a multivariate approach used to complement the cluster analysis information and to produce a dimensional scatter plot of the germplasm which is more informative regarding distances among the genotypes (Hassan et al., 2016).

Conclusion
The aim of present assay is to investigate adaptations of alien genotypes in the climate of Malakand division and Lower Dir district researched by the University of Malakand, Khyber Pakhtunkhwa. The result of our study concluded that, the accessions of H. vulgare L. exotic gene pools shows great genetic diversity within their agro-morphometric parameters and also within total seed storage protein profile assay detected through polyacrylamide gel electrophoresis SDS-PAGE technology. The diverse genetic population provides maximum opportunity for cereal breeders to select persistent, genomic insistent genotype/accession. Genetic diversity was assessed in this study through polyacrylamide base SDS-PAGE technique, which shows that the greatest genetic diversity was in loci/band number B26 (0.98%) and the least diversity was observed in loci number three (B03 = 0.16). After evaluating and sorting the gene pool of 198 exotic accessions, 40 accessions were considered as novel accessions from China, Japan and America, because these accessions are unique in their qualitative and yield contributing traits. These forty accessions also reproduce 26 protein bands markers on the electropherogram of the gel electrophoresis technique. The present study also revealed that SDS-PAGE is not only helpful for assessment of genetic diversity. DNA base markers and other spectrometric based techniques and multivariate agro-climate trials are needed for further assessment to find and conserve the gene pool index and enhance genetic diversity for crop improvement, which is needed in the context of climate changes and germplasm conservation.