Genetic variations of cultivars are very interesting in reducing genetic vulnerability and lead to stable control of production. The aim of this research was to study genetic diversity among six durum wheat cultivars. For the first assay we evaluated seven morphological traits which are: spikelet per spike, spike length, spike width, beard length, plant height, width of truncation and barb length. The tested genotypes were classified in three groups according to the linkage distance analysis. The genetic variability was also evaluated for seed storage-proteins by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoregram allowed the estimation of the durum wheat genetic similarity (GS). This GS analysis based on Unweighted Pair Group Method with Arithmetic averages (UPGMA), permits to obtain the same genotypic clustering. No significant correlation was observed among the two methods tested. It is concluded that seed storage protein profiles could be useful markers in the studies of genetic diversity and genotypes classification, which can be used to improve the efficiency of wheat breeding programs.Key words: Wheat genotypes, SDS-PAGE, genetic diversity, cluster analysis

Amara, Slim H.

Ayed, S.

Karmous, C.

Slim, A.

English

AJOL - African Journals Online

African Journal of Biotechnology Vol. 9(49), pp. 8277-8282, 6 December, 2010     Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB09.1862 ISSN 1684–5315 © 2010 Academic Journals     Full Length Research Paper  Genetic variation of durum wheat landraces using morphological and protein markers  S.Ayed1, C. Karmous2, A. Slim1 and H. Slim Amara1  1National Institute of Agriculture of Tunis, 43, Avenue Charles Nicole, 1082 Tunis, Tunisia. 2Department of Plant Sciences, Agricultural High School of Mateur, Route de Tabarka, 7030, Mateur, Tunisia.  Accepted 3 June, 2010  Genetic variations of cultivars are very interesting in reducing genetic vulnerability and lead to stable control of production. The aim of this research was to study genetic diversity among six durum wheat cultivars. For the first assay we evaluated seven morphological traits which are: spikelet per spike, spike length, spike width, beard length, plant height, width of truncation and barb length. The tested genotypes were classified in three groups according to the linkage distance analysis. The genetic variability was also evaluated for seed storage-proteins by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoregram allowed the estimation of the durum wheat genetic similarity (GS). This GS analysis based on Unweighted Pair Group Method with Arithmetic averages (UPGMA), permits to obtain the same genotypic clustering. No significant correlation was observed among the two methods tested. It is concluded that seed storage protein profiles could be useful markers in the studies of genetic diversity and genotypes classification, which can be used to improve the efficiency of wheat breeding programs.  Key words: Wheat genotypes, SDS-PAGE, genetic diversity, cluster analysis.   INTRODUCTION  Wheat genetic diversity has been well evaluated using morphological protein and molecular markers. Phenotypic identification based on morphological traits has been successfully used for genetic diversity analysis (Daâloul et al., 1998; Fakhfak et al., 1998). However, morpho-logical traits have a number of limitations, including low polymorphism, low heritability, late expression, and may be controlled by epistatic and pleiotropic gene effects (Nakamura, 2001). While protein markers, like seed storage proteins, reflect with more accuracy the genotypes, independently from the environmental effects (Autran et al., 1995), protein markers are useful tools in identifying cultivar, registration of new varieties, classification of crop    *Corresponding author. E-mail: ayedsourour@yahoo.fr. Tel: 21696285244. Fax: 216 71 799 391.  Abbreviations: MWM, Molecular weight marker; LWM, Low molecular weight; HWM, high molecular weight; HWMGS, high molecular weight glutenin subunits; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; GS, genetic similarity.  species and in studying genetic diversity, thereby improving the efficiency of wheat breeding programs in cultivar development (Gianibelli et al., 2001; Naghavi et al., 2009).  Proteins are grouped into four classes according to their solubility: albumins, globulins, prolamins and glutenins. According to the proteins quantity and quality, mainly gluten, wheat varieties are regrouped in different classes (Godon and Willm, 1991). Gluten, comprising roughly 78 - 85% of total wheat endosperm protein, is a very large complex composed mainly of polymerics and proteins known respectively as glutenins and gliadins (Mac Ritchie, 1994). Gliadins and glutenins have been extensively studied and the genetics and biochemistry are relatively well known (Magdalena et al., 2002; Starovicova et al., 2003; Picard et al., 2005). Glutenins confer elasticity to dough, whereas, gliadins are viscous and give extensibility to dough (Feillet, 2000).  The main objective of this study was to study genetic diversity in landraces of durum wheat using morphological data and seed storage proteins. This information will be useful to improve techniques for sampling wheat genetic variation which might  increase  efficiency  of  germplasm  8278         Afr. J. Biotechnol.    Table 1. Variance analysis (Mean squares and test F) for seven traits for six (6) durum wheat landraces.  Variation sources df Plant height Spike length Spike width Barb length Truncation width Beard length Spiklet per spike Genotypes 5 2623.17** 9.24** 0.11** 15.52** 3.91** 1.73** 9.24** CV (%)  18.8 9.2 18.4 20.8 18.4 16.7 25.3 R2  0.87 0.50 0.73 0.64 0.78 0.91 0.90    Table 2. Mean values of different traits in six durum wheat genotypes.   Parameters Hamira 1 Hamira 2 Hamira 3 Agili RC1 Agili RC2 Agili RP1 Plant height (cm) 84.60 b1) 126.00 c 120.00 a 135.80 d 131.40 cd 138.60 d Spike length (cm) 8.50b 6.68 a 6.28 a 9.74 b 9.80 ab 9.20ab Spike width (cm) 1.08bc 0.64a 0.60a 0.80d 0.88b 0.74cd Barb length (cm) 10.00b 8.22a 8.04c 12.12b 11.40b 12.32ab Beard length (mm) 2.50c 3.00d 2.90d 1.80a 2.00b 1.90b Truncation width (mm) 5.40ab 6.20ab 6.00a 4.80c 4.90b 4.60c Spikelet per spike 25.00c 19.80d 19.20d 23.40a 22.00b 22.00b  Values by same letters in each row are not significantly different at the 5% probability level by Duncan’s multiple.    conservation.   MATERIALS AND METHODS  Plant materials  Six Tunisian landraces of durum wheat (Triticum turgidum L.var. durum (Desf.)) were used in this study (Hamira 1, Hamira 2, Hamira 3, Agili RC1, Agili RC 2 et Agili glabre RP1). These genotypes were evaluated with five replications at the experimental station of National Agronomic Institute. Each genotype was sown in a 1 m long row with 0.5 m row spacing. Morphological data on spikelet per spike, spike length, spike width, beard length, plant height, width of truncation and barb length were recorded from five plants which had been randomly chosen in each row and mean of quantitative data sets were used for analysis.    SDS PAGE electrophoresis  The seeds were crushed finely and the flour was mixed in an extraction buffer of 0.125 M Tris-Hcl (pH 6.8), 3% sodium dodecyl sulfate (SDS), 0.03% bromophenol blue and 5% 2-mercapto-ethanol. Samples were boiled for 2 min at 100°C and then centrifuged for 5 min at 10400 rpm before being fractionated by SDS-PAGE.  According to Lammeli (1970) method, a stacking gel containing 30% acrylamide, 1% Bis acrylamide, 0.4% SDS and 0.5 M Tris-Hcl (pH = 6.8), and separating gel containing 30% acrylamide, 1% Bis acrylamide, 0.4% SDS and 1.5 M Tris-Hcl (pH 8.8) were used. The two gels were polymerised in the presence of TEMED and 10% ammonium persulfate. Gels were stained overnight with 0.2% comassie Brilliant Blue and then distained overnight in water.   Estimates of genetic similarity  Genetics   similarity  (GS)  between  two  genotypes  A  and  B  was calculated for each marker system and across marker systems according to the formula (Nei and Li, 1979):  GSAB = 2NAB/ (NA+NB)   where NAB is the number of bands in common mobility, and NA and NB are the total number of bands in genotype A and B. Thus GSAB reflects the proportion of bands in common between two genotypes and may range from 0 (no bands) to 1 (identical profiles of two lines).   Cluster analysis  The similarity matrices obtained with the two sets of data (morpho-logical and biochemical level) were converted to dissimilarity matrix (d) using a formula d = 1 - GS and used to generate unweighted pair group method with arithmetic averages (UPGMA) dendro-grams. All recorded data were analyzed for variance analysis (ANOVA) and to establish dendrograms using the computer software package SPSS and STATISTICA.   RESULTS  Morphological diversity evaluation  Analysis of variance showed a significant difference between the 6 durum wheat genotypes on the morpho-logical level for the seven traits (Table 1).  Analysis of Dancun’s multiple range tests showed that, plant height ranged between 84.6 cm for Hamira 1 and 138.6 cm for Agili RP1; landrace genotype was relatively taller in size (Table 2). Variation was observed between genotypes for spike length, spike width, barb length and spikelet   per   spike.   Differences   were   also  noted  on Ayed et al.        8279                     Figure1. Different shapes of truncation and bread observed for the six durum wheat genotype. (a) Hamira 1; (b) Hamira 2; (c) Hamira 3; (d) Agili RC2; (e) Agili RC1; and ( f) Agili RP1.    Table 3. Coefficient matrix of six wheat varieties based on morphological traits.  Genotypes Hamira 1 Hamira 2 Hamira 3 Agili RC1 Agili RC2 Agili RP1 Hamira 1 0.0      Hamira 2 41.8 0.0     Hamira 3 36.0 6.1 0.0    Agili RC1 51.3 11.7 17.3 0.0   Agili RC2 46.9 7.5 12.8 4.7 0.0  Agili RP1 54.1 13.8 19.6 3.2 7.3 0.0    truncation width and beard length (Figure 1).  The dendrogram calculated from the similarity coefficient (Table 3) and unweighted pair group method with averages on the seven measured traits, is presented in Figure 2.    Genetic diversity evaluation  The six (6) durum wheat genotypes used in the present study showed various banding pattern using SDS-PAGE technique. In this study, SDS-PAGE of grain proteins was performed in order to investigate genetic diversity among wheat genotypes. Electrophoregram showing protein banding pattern of different wheat varieties are given in Figure 3. The presence or absence of bands is mentioned in Table 4. The genetic similarity coefficient matrix (Table 5) of six durum wheat genotypes on the bases of linkage distances (Euclidian distances) was used to construct a dendrogram, to find the diversity among given durum wheat genotypes (Figure 4). The study of correlation coefficient (r = 0.30) between morphological and biochemical dendrogram showed that no significant correlation was observed between the two dendrograms obtained.   DISCUSSION   Variation among six local durum wheat accessions was highly significant (p < 0.01) for all of the seven tested characters. In fact, high variability in Tunisian durum wheat was already noted by Gashaw et al. (2007) and Fakhfak et al. (1998). On the basis of morphological traits cluster analysis, placed the six  durum  wheat  genotypes  8280         Afr. J. Biotechnol.      Figure 2. UPGMA cluster analysis showing the diversity and relationship among 6 durum wheat genotypes based on seven morphological traits.                                                 H1 H2 H3 ARC1 ARC2 ARP1   MWM 116 KD    97.4KD 64 KD 16 KD   Figure 3. Electrophoregram showing protein banding pattern of different durum wheat genotypes (MWM: Molecular weight marker; H1: Hamira 1; H2: Hamira 2; H3: Hamira 3; ARC1: Agili RC1; ARC2: Agili RC2; APR1: Agili RP1).    into different groups. At Euclidean distance of 10, all genotypes showed similarity with one another and constitute two categories, one containing Hamira 1. The second formed by the rest of genotypes. At Euclidean distance of 5, the dendrogram revealed three main groups. The first contained Hamira 2, Hamira 3. The second was Agili RC1, Agili RC2, and Agili RP1. The genotype Hamira 1 was in another group. Same finding by Naghavi et al. (2009) based on morphological data (spiklet per spike, seed per spike, 100 grain weight, plant height, peduncle length  and spike length) showed that the first two principal component scores separated Iranian landraces from cultivars showing that the studied cultivars are quite different from Iranian landraces. According to the results of the SDS-PAGE, a total of 14 bands were obtained bands number 7, 9 and 13 are common in all genotypes but other bands showed variation. Agili RC1 is characterized by the presence of band 11. The genotype Agili RC2 and Agili glabre RP1 differed by the presence of bands 1 and 2 observed at Agili glabre RP1. Indeed, Hamira 1 was characterized by the presence of bands 3 and 14 but Hamira 3 presented bands 11 and 14. These bands did not exist at Hamira 1 which was characterized by the presence of the band 12. The genetic dissimilarity dendrogram calculated from similarity coefficient for LMW and HMW glutenin subunits bands revealed different groups. At linkage distance of 1.6, genotypes distributed into three groups. The first containing Hamira 2 and Hamira 3. The second was Agili RC1, Agili RC2, and Agili RP1. The genotype Hamira 1 was in another group. Thus, a large variation between genotype was noted. Several authors studied the polymorphism as well as proteins’ different mobilities via the SDS-PAGE electrophoresis (Cherdouh et al., 2005; Carello et al., 2006). Indeed, Benmoussa et al. (2000) showed large variation among LMW and HMW glutenin subunits. It has been suggested that deletions and insertions within the repetitive regions are responsible for these variations in length. Moreover, Naghavi et al. (2009) reported that most of  landraces  showed  different  Ayed et al.        8281    Table 4. Matrix of presence or absence of bands of different durum wheat genotypes.  Band Hamira 1 Hamira 2 Hamira 3 Agili RC1 Agili RC2 Agili RP1 Band 1 1 1 1 1 0 1 Band 2 1 1 1 1 0 1 Band 3 0 1 0 0 0 0 Band 4 1 1 1 0 0 0 Band 5 1 1 1 0 0 0 Band 6 1 1 1 0 0 0 Band 7 1 1 1 1 1 1 Band 8 0 0 0 1 1 1 Band 9 1 1 1 1 1 1 Band 10 1 1 1 0 0 0 Band 11 0 0 1 1 0 0 Band 12 1 0 0 0 0 0 Band 13 1 1 1 1 1 1 Band 14 0 1 1 1 1 1    Table 5. Coefficient matrix of six durum wheat genotypes based on SDS-PAGE using UPGMA method.  Genotypes Hamira 1 Hamira 2 Hamira 3 Agili RC1 Agili RC2 Agili RP1 Hamira 1 0.00      Hamira 2 1.73 0.00     Hamira 3 1.73 1.41 0.00    Agili RC1 2.83 2.65 2.24 0.00   Agili RC2 3.00 2.83 2.83 1.73 0.00  Agili RP1 2.65 2.45 2.45 1.00 1.41 0.00      Figure 4. UPGMA cluster analysis showing the diversity and relationship among 6 durum wheat genotypes based on SDS-PAGE. 8282         Afr. J. Biotechnol.    HMWGS compositions compared with cultivars. These differences may be due to the dissimilarity of materials and/or the fact that these cultivars are not originated from Iranian landraces. The variation in high molecular weight protein subunits could be the result of gene silencing in some varieties.  However, other studies showed low degree of hetero-geneity between wheat genotypes tested (Lawrence and Shephred, 1980; Mohd et al., 2007; Siddiqui and Naz, 2009). This low level of genetic diversity may be attributed to reduce number of varieties used for wheat cropping. No significant correlation was observed in the present study between the morphological and biochemical dendrogram. This result was also obtained by using morphological traits and SDS-PAGE electro-phoresis on 30 ancestrals to modern hard red winter wheat (Triticum aestivum L.) cultivars. A large variation was observed in the morphological and biochemical level between durum wheat genotypes. These two methods could be used to study genetic diversity in different durum wheat genotypes. The choice of the method for genetic diversity estimation depends largely upon the tools available and how well it fits in breeding scheme. Both biochemical and agronomical traits will be useful to breeders to formulate crosses by choosing genotypes with appro-priate characters. SDS-PAGE is widely used due to its simplicity and effectiveness for describing the genetic structure of crop germplasm.    REFERENCES  Autran J, Pogna N, Kurdryvtesv A (1995). Use of genetic variation in improvement of quality in durum wheat quality in the Mediterranean region. Options méditerranéennes Série A : Séminaires Méditerranéens, 22: 173-180. Benmoussa M, Vézina LP, Pagé M , Yelle S, Laberge S (2000). Genetic polymorphism in low-molecular-weight glutenin genes from Triticum aestivum, variety Chinese Spring, 2000. Theor. Appl. Genet. 100: p. 5. Carello JM, Vazquez JF, Orkellana J (2006). Relationship between Gluten Strength and Glutenin Proteins in Durum Wheat Cultivars. Plant Breed. 104: 325-333. Cherdouh A, Khelifi D, Carrillo JM, Nieto-Taladriz MT (2005). The high and low molecular weight glutenin subunit polymorphism of Algerian durum wheat landraces and old cultivars. Plant Breed. 124: 338-342.  Daâloul A, Harrabi M, Amara H, Gougjil S (1998). Evaluation de la collection nationale de blé dur. Revue de l’Institut National Agronomique de Tunisie numéro spécial I.S.S.N. 0330-8065 : 337-358.        Fakhfak M, Daaloul A, Rezgui S, Yahyaoui A (1998). Evaluation des associations entre le rendement en grain et les caractères morpho- physiologiques chez le blé dur dans les régions semi-arides. Revue de l’Institut National Agronomique De Tunisie, 13: 43-51. Feillet P (2000). Le grain de blé : composition et utilisation. Edition INRA, France, p. 308.  Gashaw A, Mohammed H, Singh H (2007). Selection criterion for improved grain yields in Ethiopian durum wheat genotypes. Afr. Crop Sci. J. 15: 25-31. Gianibelli MC, Gupta RB, Lafiandra D, Margiotta B, MacRitchie F (2001). Polymorphism of High Mr Glutenin Subunits in Triticum tauschii: Characterisation by Chromatography and Electrophoretic Methods J. Cereal Sci. 33: 39-52. Godon B, Willm C (1991). Les industries de la première transformation des céréales. Techniques et documentation Edition Lavoisier, p. 315. Lawrence GJ, Shepherd KW (1980). Variation in glutenin protein Subunits of wheat. Aust. J. Biol. Sci. 33: 221-233. Mac Ritchie F (1994). Role of Polymeric Proteins in Flour Functionality. In Wheat kernel proteins: molecular and functional aspects. Bitervo, Italy, Universita degli studi Della Tuscia. pp. 145-150. Magdalena R, Rodriguez M, Metakovsky V, Vazquez J, Carrillo M (2002). Polymorphism variation and genetic identity of Spanish common wheat germplasm based on gliadin alleles. Field Crops Res. pp. 185-196. Mohd S, Alam Z, Zahir A, Waqar A, Taufiq A, Ikhtiar K (2007). Characterization of wheat varieties by seed storage protein electrophoresis. Afr. J. Biotechnol. 6: 497-500. Naghavi MR, Rashidi Monfared S, Ahkami AH, Ombidbakhsh MA (2009). Genetic Variation of Durum Wheat Landraces and Cultivars Using Morphological and Protein Markers. Proceedings of world academy of science, Eng. Technol. Vol. 37. Nakamura H (2001). Genetic diversity of high-molecular-weight glutenin subunit compositions in landraces of hexaploid wheat from Japan. Euphytica, 120: 227-234. Picard P, Bourgoin GM, Zivy M (2005). Two-Dimensional Electrophoresis Potential of two-dimensional electrophoresis in routine identification of closely related durum wheat lines. Electrophoresis, 18: 1174-1181. Siddiqui MF, Naz N (2009). Protein landmarks for diversity assessment in wheat genotypes. Afr. J. Biotechnol. 8: 1855-1859. Starovicova M, Galova Z, Knoblochova HG (2003). Identification of Glutenin Markers in Cultivars of three Wheat Species. Plant Breed. 39: 51-57.                 

Genetic variation of durum wheat landraces using morphological and protein markers

https://www.ajol.info/index.php/ajb/article/download/130721/120298

Genetic variation of durum wheat landraces using morphological and protein markers

Abstract

Similar works

Full text

Available Versions

AJOL - African Journals Online