Many studies were conducted to determine the composition of LMW & HMW glutenin subunits in bread wheat (Jin et al. 2011; Atanasova et al. 2009; Henkrar et al. 2017). Their appropriate composition controls the quantity and quality of the gluten protein and is crucial in determining the rheological properties of the dough. The characterization of LMW & HMW glutenins subunits in bread wheat is crucial for developing genotypes with desired bread quality. SDS-PAGE was used in earlier investigations to analyze the composition of HMW and LMW-GS. However, because of its complexity and the similarity in electrophoretic mobility between gliadins and LMW-GS, this approach is not appropriate for high-throughput study (Liu et al. 2008, Gupta et al. 1994; Masci et al. 1998; Maruyama-Funatsuki et al. 2004). For the Glu-1 and Glu-3 loci, numerous thorough genomic studies have been described in recent studies. In recent studies, a lot of comprehensive genomic studies have been reported for characterizing the Glu-1 and Glu-3 loci. To study these, gene/allele-specific DNA markers were developed for HMW & LMW-GS for improving bread quality of wheat (Ahmad 2000; Radovanovic et al. 2002; Radovanovic et al. 2003; Ma et al. 2003; Lei et al. 2006; Wang et al. 2010). Furthermore, Kuchel et al. (2007) also reported the use of MAS (Marker-Assisted Selection) approach to improve the bread or dough-related properties of wheat.
The PCR results indicated that all the genotypes except Uaf-9515 and M.H-2 carried Ax2* allele and demonstrated an allelic frequency of 66.67% at Glu-A1 locus. Moreover, the absence of Ax1, Ax2 and Ax2* alleles in Uaf-9515 and M.H-21 may indicate the presence of AxNull or any other type of allele at Glu-A1 locu. There is need to investigate the that which alleles is present in Uaf-9515 and M.H-21 genotypes. Similar findings were also reported by Nucia et al. (2019), who analysed 79 genotypes of spring wheat from all around the Europe and found that Ax2* had highest frequency (71%) at Glu-A1 locus. Jin et al. (2011), who evaluated 719 wheat genotypes from 20 countries to assess the composition of HMW-GS, complement the findings of the current study by demonstrating that Ax2* had a 43.3% allelic frequency among the studied genotypes. These studies also confirmed that the presence of Ax2* allele had strong influence on dough and bread related properties. Moreover, our PCR findings for Glu-B1 locus indicated the high variation and confirmed the presence of Bx7 and Bx7* alleles in the studied genotypes except from Uaf-9515, Dilkash-20 and M.H-21. These genotypes might contain unidentified or other alleles like Bx17, Bx7OE, and Bx6. The presence of Bx7 and Bx7* showed an allelic frequency of 44.45 and 33.33%, respectively. Our findings conflict with prior research by Nucia et al. (2019), and Jin et al. (2011). They discovered that in the European wheat germplasm, Bx7* is the allele that is more prevalent than Bx7. Similarly, Esp' et al. (2013) and Henkrar et al. (2017) also noted a higher frequency of allele Bx7*. However, our PCR results for "y" type glutenin subunits indicated that By18 allele was absent and showed 0% allelic frequency, but By8 allele showed 66.67% allelic frequency due to its presence in all genotypes apart from Uaf-10136, Subhani-21, and M.H-21 at Glu-B1, as shown in Tables 2 & 3. These genotypes may carry the By9, Bynull, and By8* alleles. Therefore, it is necessary to identify the unknown alleles utilizing a variety of techniques, such as peptide mass sequencing and nucleotide sequencing. In contrast to the current work, Janni et al. (2017) identified 19 genotypes of bread and durum wheat and noted that the By8 allele had just 16% allelic frequency. Jin et al. (2011) also found that By8 had a higher allelic frequency (31.1%) than By9, which had only a 22.28% allelic frequency. In the present study, By8 also showed higher frequency but its absence in some genotypes concluded that higher variability is present at Glu-B1 locus in bread wheat.
At Glu-D1, a single allelic combination Dx5 + Dy10 was observed in all the genotypes and showed 100% allele frequency. This indicated that both Dx5 and Dy10 alleles are more common in Pakistani spring wheat genotypes. A similar study was conducted by Ali et al. (2013), who also confirmed the high allelic frequency (95%) of Dx5 + Dy10 combination in Pakistani spring wheat genotypes. Moreover, Dias et al. (2017) and Henkrar et al. (2017) also confirmed the high allelic frequency of Dx5 + Dy10 in their studies that was 73% and 85%, respectively. Nucia et al. (2019) also observed that more than 80% European’s wheat genotypes had Dx5 + Dy10 allele at Glu-D1 locus. This allele showed a strong association with good bread-making properties as compared to Dx2 + Dy12 allelic combination (Costa et al. 2013 and Barakat et al. 2018).
At Glu-A3 locus, only a few genotypes showed the presence of gluA3b allele and an allelic frequency of 55.56%. This lower frequency %age may point out the presence of other alleles such as the a and g allele at the Glu-A3 locus. Similar results were reported by Zhang et al. (2004), who discovered six distinct allelic variants of a single gene encoding LMW glutenin and developed six allele-specific markers to differentiate each allele from the others. Similarly, the presence of gluB3b, gluB3c, and other unknown alleles at the Glu-B3 locus also showed the existence of several other LMW glutenin alleles. Additionally, the allelic frequencies for the gluB3b and gluB3c alleles were 77.78 and 11%, respectively as shown in Tables 2 & 3. The observed differences in allelic frequency also evident the presence of several LMW glutenin alleles at the Glu-B3 locu. Different alleles of the Glu-B3 locus, including alleles a, b, c, d, e, f, g, h, and I were also found by Gupta and Shepherd (1990). Additionally, Costa et al. (2013) discovered many alleles and revealed that the spring wheat genotypes under study had the highest frequency of gluB3b (33.33%). Our findings support earlier research, which reported the significant level of polymorphism at the Glu-B3 locus.
Molecular findings were further validated with various bread quality-related tests such as farinograph, extensograph, sedimentation, and bread volume. These analytical tests proved helpful in assessing the effectiveness of Marker-Assisted Selection (MAS) technique that was utilized in this study for genotyping and selection of wheat genotypes that contributed to good bread-making properties. The farinographic analysis indicated that, genotype Uaf-10137 absorbed more water than Uaf-9515 by a wide margin. Similar findings were reported by Simon (1987), who concluded that greater water absorption is a sign of high-quality flour and is necessary for baking bread of excellent quality. Other genotypes, on the other hand, displayed non-significant differences for WA%; this could be because various alleles have a similar effect or because other flour quality parameters play an important role. Future research on this figure will require a thorough comprehension of him.
Additionally, the dough development time data demonstrated that all genotypes, except for Subhani-21, Uaf-9515, and M.H-21 genotypes, showed largest and non-significant differences for DDT. These three genotypes might exhibit less DDT because of the different HMW and LMW glutenin subunits. Stronger flour is typically indicated by a longer dough development time, whereas a lower value denotes weaker flour. Safdar et al. (2009) also reported that genotypes with higher DDT indicated good quality flour as compared to genotypes with lower DDT. In addition to this, Uaf-10137 genotype indicated highest DST (Dough Stability Time) and lowest MTI (Mixing Tolerance Index) or dough softening whereas Uaf-9515 had lowest value for DST and highest MTI value. The higher DST and lower MTI values also indicate strong flour quality, and these rheological properties are required to make good bread (Anjum and Walker 2000). Moreover, all the genotypes showed a minor difference for both DST and MTI values in the present study, it may be due to the minor differences in the protein quality and quantity in the studied genotypes. Similar findings were also reported in many studies (Rehman et al. 2001).
The Extensograph results indicated that Uaf-10137 had highest RE (Resistance to Extension) and E (Extensibility). However, Subhani-21 and M.H-21 had lower RE and E, respectively. Our results suggested that genotypes had varying performance for both RE and E parameters of Extensograph. It implies that these two factors alone are insufficient to ascertain the rheological or viscoelastic characteristics of the dough. Moreover, the differences in the Extensographic properties may be exhibited due to the presence of different allelic combinations at both Glu-1 and Glu-3 loci. Torbica et al. (2007) concluded that the differences in RE and E may occur due to imperfect balance between gliadin and glutenin content. The imperfect balance between these two proteins leads to increased extensibility whereas lower resistance to extension. Torbica et al. (2011) also observed that RE and E parameters of Extensograph are not enough to determine the visco-elastics properties of the bread. Now there is a need to study other parameters of Extensgraph while selecting genotypes for good visco-elastics properties.
The SDS-Sedimentation volume is widely used to measure the gluten quality and its strength. Additionally, it also gives an information about bread-making properties. The findings of the sedimentation test revealed that Uaf-10137 had the maximum volume of sedimentation while Uaf-9515 had the lowest amount. A significant positive correlation between loaf volume and sedimentation volume was found by Guzmán et al. (2022). Moreover, they also found that Glu-D1 locus had less 1% contribution to this character. However, at Glu-3 locus, gluA3b and gluB3b alleles were also showing a strong association with sedimentation volume. Our results are also in agreement with Guzmán et al. (2022). Our findings showed a substantial difference between the genotypes for bread volume. The genotype Uaf-10137 displayed the highest value for bread volume, whereas the genotype Uaf-9515 displayed the lowest value. These significant variations could be the result of distinct HMW and LMW glutenin subunits. Similar results were reported by Guzmán et al. (2022). They concluded that environmental conditions and protein content, in addition to the makeup of different alleles, also affect bread volume. They also confirmed that gluA3b and gluB3b alleles had higher contributions to bread volume than Glu-D1, which has a minimal effect on bread volume. These findings concur with the current investigation.