Acta agriculturae Slovenica, 117/2, 1–8, Ljubljana 2021 doi:10.14720/aas.2021.117.2.2050 Original research article / izvirni znanstveni članek Gene action for grain protein content in durum wheat Rangel DRAGOV Received January 16, 2021; accepted April 25, 2021. Delo je prispelo 16. januarja 2020, sprejeto 25. aprila 2021. Gene action for grain protein content in durum wheat Abstract: The aim of this study was to determine the gene action and combining ability of durum wheat for grain protein content. During the three year period a diallel cross was carried out with five modern parents of durum wheat – ‘Victoria’, ‘Deni’, ‘Superdur’, ‘Progres’ and ‘Predel’. Ten hybrid combinations and the parents were grown in the experimen- tal field of the Field Crops Institute, Chirpan. The experiment was performed by the randomize block method design in three replications. It was found that in the inheritance of grain pro- tein content dominance and overdominance in positive and negative directions were observed. Statistical processing of the results showed that both additive and non-additive genetic ef- fects have influenced on inheritance. Non-additive gene effects (SCA) had a greater role in inheritance. This suggests that an effective selection for this trait could begin in later generations. The combining ability analysis has identified two good general combinators (Predel and Superdur varieties) that could be used as donors to increase the values of the trait protein content in grain. Several crosses showing positive and significant SCA ef- fects have also been identified, suitable for achieving reliable transgressive genotypes. Key words: gene action; combining ability; GCA effects; SCA effects; grain protein content; durum wheat Field Crops Institute, Chirpan, Bulgaria Corresponding author: dragov1@abv.bg Delovanje genov za vsebnost beljakovin v zrnih trde (du- rum) pšenice Namen raziskave je bil določiti vpliv delovanja genov in kombinacijske sposobnosti trde (durum) pšenice na vsebnost beljakovin v zrnih. V obdobju treh let so bila izvedena dialelna križanja s petimi sodobnimi starševskimi sortami trde pšeni- ce-– Victoria, Deni, Superdur, Progres in Predel. Deset križan- cev in njihovi starši so bili gojeni na poskusnem polju Inštituta za poljščine v Čirpanu (Field Crops Institute, Chirpan). Poskus je bil izveden kot naključni bločni poskus s tremi ponovitvami. Ugotovljeno je bilo, da sta pri dedovanju vsebnosti beljakovin v zrnju udeležni dominanca in naddominanca v pozitivni in ne- gativni smeri. Statistična obdelava podatkov je pokazala, da so na dedovanje vplivali aditivni in neaditivni genski vplivi. Nea- ditivni genski učinki so imeli pri dedovanju večjo vlogo. To na- kazuje, da bi se učinkovita selekcija za to lastnost lahko začela že v prejšnjih generacijah. Z analizo kombinacijske sposobnosti sta bila prepoznana dva dobra splošna kombinatorja (‘Predel’ in ‘Superdur’), ki bi ju lahko uporabili kot donatorja za povečanje vsebnosti beljakovin v zrnih. Številna križanja kažejo pozitivne in značilne neaditivne genske učinke (SCA), ki so primerni za doseganje zanesljivih transgresivnih genotipov. Ključne besede: delovanje genov; kombinacijska sposob- nost; aditivni genski učinki; neaditivni genski učinki; vsebnost beljakovin v zrnju; trda (durum) pšenica Acta agriculturae Slovenica, 117/2 – 2021 2 R. DRAGOV 1 INTRODUCTION Durum or pasta wheat is the only tetraploid species of commercial importance that is cultivated in a number of countries, and in some of them it is essential. Durum wheat is an important crop for the preparation of pasta, bulgur, couscous and other products (Ahmad, 2015). The protein content of the grain is of particular importance for the quality of durum wheat and its products. High values of this trait have a positive relationship with the yield of semolina from the grain and are desired by the milling industry. The use of plant proteins in human food is of great importance for healthy human nutrition. The protein content in the grain is a very important indicator of wheat related to its nutritional value and technologi- cal qualities of the products (Blanco et al., 2006). Protein content is highly influenced by the genotype and content of available nitrogen in the soil, as well as humidity and temperature during the growing season (Campbell et al., 1977; Fowler et al., 1990; Ames et al., 2003). According to Clarke et al. (1990) and Fowler et al. (1990) the highest protein content of the grain is due to the cultivation of varieties that can withstand higher rates of nitrogen ferti- lization. The improvement of the varieties must meet the requirements for the production of high quality products. This leads to an accelerated improvement of individual traits. Knowledge of the gene action of the trait is useful for the development of a high quality durum wheat vari- ety. Diallel crosses are used to establish gene action and mechanisms for inheriting quantitative traits. It is a reli- able method for selecting parents and providing a com- prehensive assessment of their hybrid combinations. This analysis is suitable for studying quantitative traits in early generations. It requires relatively more work, but gives a very effective estimate. More detailed information can be obtained from diallel crosses with one generation F 1 . The other important advantage is that the estimates obtained in F 1 can be confirmed by testing the second generation F 2 . In diallel analysis, general combining ability (GCA) is associated with genes that have additive effects and describes the ability of parents to pass on their traits to hybrid generation. Specific combining ability (SCA) is the deviation from additive effects caused by dominance and epistasis. The establishment of gene action (additive and non-additive) is of great importance for determin- ing the strategy for achieving more meaningful and rapid results in the development of new varieties. In a properly designed breeding program, knowledge of gene action is the key that will maximize the effectiveness of improve- ment breeding work. A number of authors have studied gene action in inheritance of grain protein content in wheat. Lysa (2009), Akram et al. (2011), Yao et al. (2014), Pansuriya et al. (2014), Tiwari et al. (2015) have reported significant participation of both additive (GCA) and non-additive (SCA) gene effects in the inheritance of the studied trait. According to the ratio of GCA / SCA variance, additive gene effects (Ϭ g 2 > Ϭ s 2 ) have played a greater role in inher- itance. This suggests that this trait could be significantly more easily improved and it is possible for an effective selection of genotypes to be applied in earlier segregated generations. Other researchers (El-Habbad et al., 1996; Pansuriya et al., 2014; Tiwari et al., 2015) found prepon- derance of non-additive genetic effects, while Zahid et al. (2007) and Al-Naggar et al. (2015) noted the preponder- ance of additive gene effects. From the preponderance of the non-additive genetic effects reported by some authors, it could be assumed that dominance had a greater influ- ence on this trait. Determining the degree of dominance would help to establish the possibility of purposeful work in the breeding program. Here, a link can be made with the use of specific results, aimed at reaching transgres- sive forms or creating heterosis varieties. According to Fonseca & Patterson (1968), Martin & Talbert (1995), Elfadl et al. (2006) dominance and overdominance cor- respond to the theoretical foundations of heterosis and suggest that it is the result of allelic and non-allelic in- teractions between the genetic material of the parents. Dominance in the inheritance of the trait was reported by Patel et al. (2018). The diversity of the results obtained is evidence of the great influence of the genotypes used and the environmental conditions in the inheritance of the trait. This suggests that modern domestic and foreign varieties of durum wheat should be included in the study and their genetic capabilities should be traced and deter- mined. Plant breeding is the art and science of changing the heredity of plants and improving them for the benefit of human. The aim of this research was to study the genetic difference between genotypes, as well as the type of gene action (additive and non-additive) in the inherit- ance of protein content in the grain. By determining the general and specific combining ability, we expected to identify the parents for successful combinations and to obtained promising genotypes in the earliest segregated generations. This will be of great benefit in optimizing the breeding process for durum wheat in terms of grain protein content. 2 MATERIAL AND METHODS The study was conducted in the experimental field of the Field crops institute in Chirpan, Bulgaria in three consecutive years (2014-2016) using the standard, local cultivation technology. The soil type is Eutric Vertisols Acta agriculturae Slovenica, 117/2 – 2021 3 Gene action for grain protein content in durum wheat (by FAO), characterized by medium organic matter (1.5- 2.4 %), with slightly acid to neutral soil reaction. The ex- periments were sown in the optimal period for durum wheat in Bulgaria in time-frame October 20-30. The genotypes heading time was in time-frame May 8-16. The plants were taken (harvested) in time-frame July 7-10 in full maturity. Meteorological conditions during the three-year period of the study were characterized by higher temperature than the multi-annual norm. The first two harvest years of 2014 and 2015 were favourable in terms of soil moisture and rainfall higher than the av- erage for many years. The third harvest year was charac- terized as the hottest and at the same time with 20 % less precipitation. A half diallel cross was performed with the including of five modern varieties of durum wheat: Victoria (BG), Deni (BG), Superdur (AT), Progres (BG), Predel (BG). The three-year period has allowed three generations of F 1 and two generations of F 2 to be grown. The parents, F 1 and F 2 hybrids were sown under field conditions by the block method design in three replications. Each parent or F 1 was sown in two rows, and each F 2 was sown in five rows; each row was 2 m long; spaces between rows were 20 cm and 5 cm between plants. In full maturity a total of 20 plants from F 1 and parents and 30 plants from F 2 were taken randomly from each replication every year. Part of the seeds were used for the sowing of F 2 . With the remaining seeds, a technological analysis was performed for estimation of grain protein content. The grain protein concentration (GPC, %) was estimated by measuring of N according to the Kjeldahl method. The following for- mula was used: Protein, % = N (% DM) x 5.7 to convert the N content to protein content (BDS ISO 20483:2014). The results obtained were statistically processed by applying the method 2 model I of Griffing (1956) with software product of Mark D. Burow and James G. Co- ors 1994 (Burow & Coors, 1994). The same program was used for the analysis of variance. The general combining ability (GCA) of the parents and the specific combining ability (SCA) of the crosses were determined. The degrees of dominance in the individual hybrid combinations were calculated according to Ognyanova (1975). On the results for mean of parents and their hybrid combination was conducted Duncan`s test for multiple comparing the means at the detected significant differences (p < 0.05) (Duncan, 1955). Statistica 10 software program was used for the two analyzes performed above. 3 RESULTS AND DISCUSSION The mean values for the trait grain protein content of the parents for the three years F 1 and both F 2 genera- tions are presented in Table 1. It can be seen that there was a significant variation in the years of study, also and significant differences between mean values. The most favourable for grain protein content was 2014, while the most unfavourable was 2015. The parents had val- ues from 13.75 % for the variety Victoria (F 1 -2015) to 18.53 % for the variety Superdur (F 1 -2014). The table also presents the average values of the hybrid combinations by years and generations. The highest value was found for the combination Victoria X Deni - 18.50 % (F 1 -2014), and the lowest - 13.52 % for Victoria X Progres (F 2 -2015). The same table includes the corresponding indices show- ing the ways and direction of inheritance. They show that there was a great diversity in inheritance. In the F 1 generation in 2014 positive overdominance (towards the better parent) prevailed. In 2015, there were more mani- festations of intermediate inheritance, but there also were those with dominance and over-dominance to the weaker parent. In 2016 dominance and overdominance in both directions were observed. In the F 2 generation, several manifestations of overdominance were seen in a positive direction, but in most cases it was in a negative direction. In inheritance of the grain protein dominance and overdominance in both positive and negative direc- tions were observed. Preponderance of dominance and overdominance for the trait grain protein content was established by other authors (Kumar & Maloo, 2011; De- sale & Mehta, 2013; Patel et al., 2018). Table 2 represents the analysis of variance for gen- otypes, general combining ability (GCA) and specific combining ability (SCA). Significant differences between genotypes were observed for all test cases. This makes it possible to conduct a diallel analysis for an in-depth study of the genetic causes controlling the trait grain protein content. The sums of the squares of the genotypes were in each case the largest and determine that the genotypes had the largest contribution to the overall variation of the studied trait. The values of the mean squares for geno- types, GCA and SCA were statistically significant for the three harvest years in both generations (table 2). There- fore, both additive gene effects (GCA) and non-additive gene effects (SCA) were involved in the inheritance of the trait. The results correspond to those obtained by a number of other authors (Barnard et al., 2002; Joshi et al., 2004; Lysa, 2009; Akram et al., 2011; Y ao et al., 2014; Pan- suriya et al., 2014; Tiwari et al., 2015; Patel et al., 2018). The ratio of GCA and SCA variances indicates a predominance of non-additive gene effects over ad- ditive ones in all cases except F 1 -2015, where additives predominated. The same results, for the predominance of non-additive genetic effects, have been reported by a number of other authors (Perenzin et al., 1992; Singh et al., 2004; Joshi et al., 2004; Nazeer et al., 2011; Kumar, Acta agriculturae Slovenica, 117/2 – 2021 4 R. DRAGOV Table 1: Mean values and indexes of inheritance for trait grain protein content (%). Mean values (in each column), followed by the same letters are not significantly different at p < 0.05 according to Duncan‘s multiple range test (DMRT). Indexes: i-intermediate, cd-complete dominance, pd-partial dominance, od-overdominance, minus- decrease, plus-increase Parents Code 2014 y. 2015 y. 2016 y. 2015 y. 2016 y. Victoria 11 14.74a 13.75ab 14.47a 13.75ab 14.47a Deni 22 16.25abc 14.67bcd 16.27cde 14.67bcd 16.27cde Superdur 33 18.53d 14.59abcd 16.43de 14.59bcd 16.43cde Progres 44 15.35a 15.04d 15.09abc 15.04cd 15.09abc Predel 55 17.70bcd 15.31d 16.21cde 15.31d 16.21cde Hybrid combinations Code F 1 -2014 y. F 1 -2015 y. F 1 -2016 y. F 2 -2015 y. F 2 -2016 y. Victoria x Deni 12 od+ 18.50d cd- 13.89ab cd- 14.55a i 14.24abc i 15.36abcd Victoria x Superdur 13 i 16.57abcd cd- 13.76ab od+ 16.78e od- 13.65ab pd+ 16.11bcde Victoria x Progres 14 od+ 16.09ab i 14.13abc od+ 15.39abcd od- 13.52a od+ 15.54abcde Victoria x Predel 15 cd+ 17.43bcd cd- 13.97ab cd+ 16.03bcde od- 13.68ab cd- 14.77ab Deni x Superdur 23 i 17.56bcd od- 13.76a od- 15.52abcd od- 14.48abcd od- 15.49abcde Deni x Progres 24 od+ 17.28bcd cd+ 15.13d od- 14.87ab od- 14.18abc od- 14.50a Deni x Predel 25 od+ 18.13cd i 15.13d od- 15.17abcd cd+ 15.18cd od- 15.41abcde Superdur x Progres 34 pd+ 17.81bcd cd+ 15.18d i 15.68abcde cd+ 14.99cd cd- 15.14abc Superdur x Predel 35 cd- 17.69bcd i 14.96cd od- 15.71abcde od- 14.40abcd od+ 16.59de Progress x Predel 45 cd+ 17.95bcd od- 14.56abcd od+ 16.03bcde od- 14.19abc od+ 16.80e M ± m 17.17±0.29 14.52±0.15 15.61±0.17 14.39±0.14 15.61±0.19 2012; Khodadadi et al., 2012; Ahmad et al., 2017; Pa- tel et al., 2018). On the other hand, the predominance of additive genetic effects in inheritance has been reported by other researchers (El-Habbad et al., 1996; Bnejdi & El-Gazzah, 2010; Sadeghi et al., 2012; Tiwari et al., 2015; Al-Naggar et al., 2015). The predominance of non-additive gene effects suggests that an effective selection in the breeding for grain protein content in durum wheat, in our set of parents, should be conducted in later segregated generations. This is associated with a reduced influence of non-additive genetic effects in later segregated generations. Table 3 represents the values for parental GCA and hybrid crosses SCA. Predel variety manifested itself as a good general combiner for increasing the grain protein content. It had positive and significant GCA values for all test cases. This variety contained more genes with addi- tive effects. Of interest was the variety Superdur, which in three of the five cases had significant and positive values of GCA. These genotypes could be successfully used in breeding of durum wheat to increase the values of the trait grain protein content. As a bad general combiner for the trait was Victoria variety showed significant negative values for all cases of research and lead to a decrease in the protein content in the hybrids obtained with it. The other parent varieties occupied an intermediate position. There was no observed clear outlined good combination of SCA effects in the hybrid crosses. In different years there was no hybrid combination with three or more sig- nificant effects in one direction. Of interest in this situa- tion was the cross ‘Victoria’ X ‘Superdur’ , which had two significant positive values for SCA. This combination was a cross between a parent with a negative GCA and a par- ent with a positive GCA. Other crosses with high SCA effects for two years were ‘Progres’ X ‘Predel’ and ‘Super- dur’ X ‘Progres‘ . Researchers Kumar & Maloo (2012) and Singh et al. (2012) reported that not all crosses with high SCA effects were obtained from the crosses of a ’Good’ X ’Good’ GCA combiner. Particularly, crosses with high SCA effects were obtained from crosses between ‘Bad’ X ‘Bad’ and ‘Bad’ X ‘Good’ general combiner. These re- searchers claimed that such manifestations were due to the involvement of dominance or epistatic gene effects. Gami et al. (2011) and Tiwari et al. (2015) have de- termined that crosses with high SCA may be more likely to be sources of transgression. Transgressive lines on a Acta agriculturae Slovenica, 117/2 – 2021 5 Gene action for grain protein content in durum wheat Table 2: ANOV A by years for Genotypes, General combining ability (GCA), Specific combining ability (SCA) and relation to vari- ances of GCA and SCA (Ϭ g 2 / Ϭ s 2 ) for grain protein content * - p ≤ 0.05; ** - p ≤ 0.01; *** - p ≤ 0.001; n.s. – no significant Ye ar Source of variation Sum of squares Mean squares Significant (*,**,***) F 1 -2014 Genotype 53.769 3.841 *** GCA 26.434 6.608 *** SCA 27.336 2.734 *** Error 29.506 1.054 Ϭ g 2 / Ϭ s 2 0.32 F 1 -2015 Genotype 14.672 1.048 *** GCA 10.023 2.506 *** SCA 4.649 0.465 *** Error 6.347 0.227 Ϭ g 2 / Ϭ s 2 1.22 F 1 -2016 Genotype 20.008 1.429 *** GCA 7.872 1.968 *** SCA 12.13 1.214 *** Error 11.713 0.418 Ϭ g 2 / Ϭ s 2 0.11 F 2 -2015 Genotype 14.091 1.007 *** GCA 8.012 2.003 *** SCA 6.079 0.608 *** Error 7.919 0.283 Ϭ g 2 / Ϭ s 2 0.6 F 2 -2016 Genotype 23.806 1.700 *** GCA 9.805 2.451 *** SCA 14.001 1.400 *** Error 15.219 0.544 Ϭ g 2 / Ϭ s 2 0.17 given trait can be a source for creating high-nutrition va- rieties of durum wheat. According to them, assessments of gene action and variation explain the genetic poten- tial of materials and contribute to breeding progress in durum wheat quality. The analysis of combining ability shows that non-additive genetic effects (dominance and epistasis) has played a major role in the inheritance of the trait grain protein content in durum wheat. Two good combiners have been identified to increase the grain protein content: ‘Predel’ and ‘Superdur’ . Furthermore as a promising combination was found the cross ‘Victoria’ X ‘Superdur’. Varieties Predel and Superdur have been defined as good general combiners by other quantita- tive characteristics in our previous studies (Dragov & Dechev, 2015; Dragov, 2017; Dragov, 2020). The study provided information on two of the most important moments in a successful breeding program - choosing parents for crossing and leading a purposeful selection on this trait. The choice of parents for cross- breeding is the basis for obtaining good results from a breeding program. Hybridization is a basic method for increasing genetic diversity and obtaining valuable geno- types in segregating generations. When choosing par- ents, the following should be taken into account: genetic distance, adaptation potential and combining ability. Greater genetic variation and the possibility of trans- gressions were obtained from crosses with higher SCA effects. In turn, crosses with high SCA effects were ob- tained by crossing parents with high GCA effects with parents with medium or low GCA effects. This indicates Acta agriculturae Slovenica, 117/2 – 2021 6 R. DRAGOV Table 3: Values for general combining ability (GCA) of parents and specific combining ability (SCA) of crosses for grain protein content * - p ≤ 0.05 ; n.s. – no significant Code 2014 y. 2015 y. 2016 y. 2015 y. 2016 y. Parents / Error ±0.31 ±0.14 ±0.19 ±0.16 ±0.22 11 Victoria -0.70* -0.55* -0.28* -0.53* -0.42* 22 Deni 0.13 n.s. 0.01 n.s. -0.14 n.s. 0.15 n.s. -0.05 n.s. 33 Superdur 0.52* -0.04 n.s. 0.40* 0.05 n.s. 0.36* 44 Progres -0.45* 0.27* -0.21* 0.08 n.s. -0.21 n.s. 55 Predel 0.50* 0.30* 0.24* 0.24* 0.33* Hybrid combinations F 1 -2014 y. F 1 -2015 y. F 1 -2016 y. F 2 -2015 y. F 2 -2016 y. Crosses / Error ±0.70 ±0.32 ±0.44 ±0.36 ±0.50 12 Victoria x Deni 1.9* -0.09 n.s. -0.62* 0.23 n.s. 0.22 n.s. 13 Victoria x Superdur -0.41 n.s. -0.16 n.s. 1.03* -0.25 n.s. 0.55* 14 Victoria x Progres 0.08 n.s. -0.11 n.s. 0.28 n.s. -0.42* 0.56* 15 Victoria x Predel 0.46 n.s. -0.30 n.s. 0.45* -0.42* -0.75* 23 Deni x Superdur -0.26 n.s. -0.73* -0.35 n.s. -0.11 n.s. -0.42 n.s. 24 Deni x Progres 0.43 n.s. 0.31 n.s. -0.37 n.s. -0.44* -0.84* 25 Deni x Predel 0.31 n.s. 0.28 n.s. -0.53* 0.38* -0.47 n.s. 34 Superdur x Progres 0.57 n.s. 0.41* -0.12 n.s. 0.46* -0.61* 35 Superdur x Predel -0.51 n.s. 0.18 n.s. -0.55* -0.28 n.s. 0.28 n.s. 45 Progress x Predel 0.72* -0.53* 0.39 n.s. -0.53* 1.07* that parents with high GCA effects should always be pre- sent in the hybridization scheme. In our set of parents in the diallel cross, two varieties: Superdur and Predel were established as the most valuable parents. The use of these varieties in the future hybridization program would show good results in segregated generations. Successful selection in the early segregated gen- erations F 2 , F 3 is suitable for traits in which inheritance control is determined by additive gene effects. It should be noted that for the studied trait there was a significant participation of the additive genetic effects in our case. An effective selection in later segregated generations should be recommended for traits in which non-additive genetic effects predominate. Therefore, an effective selec- tion on this trait should be applied in the later segregat- ed generations, when the influence of the non-additive (dominance) decreases and the additivity increases. The significant influence of additive and non-additive gene effects found in our study suggests that the use of both types of gene effects is necessary to improve the trait. In the studied trait in F 1 in one year the inheritance was mainly controlled by additive gene effects while in the other two it was mainly by non-additive ones. The selec- tion in different environmental conditions (years) would have a positive impact on breeding improvement work. This is due to the accumulation of different useful genes in different years. 4 CONCLUSIONS In the inheritance of grain protein content there was complete dominance and overdominance, both in a positive and in a negative direction. Additive and non- additive genetic effects had a significant influence on the inheritance of this trait. Therefore, to maximize the grain protein content of durum wheat, a system that includes both types of gene effects at the same time should be used. The ratio of variances indicates that non-additive genetic effects prevailed over additive ones in most cases. This result shows that it is necessary for an effective se- lection to start in the later segregated generations where dominance decreases and additivity increases. The study identified two good combiners that increased the values of the trait: Predel and Superdur varieties. Crosses with these two genotypes suggest opportunities for promising Acta agriculturae Slovenica, 117/2 – 2021 7 Gene action for grain protein content in durum wheat hybrids. The hybrid combination ‘Victoria’ X ‘Superdur’ is also of selection interest according to the demonstrated significant values for SCA. 5 REFERENCES Ahmad, E., Akhtar, M., Badoni, S., & Jaiswal, P. (2017). Com- bining ability studies for seed yield related attributes and quality parameters in bread wheat (Triticum aestivum L.). Journal of Genetics, Genomics & Plant Breeding, 1(1), 21-27. http://ejggpb.com/abstract.php?article_id=4 Ahmad, M. (2015). Genetical analysis of some agronomic traits in durum wheat. 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