Electrophoretic variations in polypeptide patterns in total seed storage protein
The total seed protein extracts of 34 Indian barley lines were separated on SDS-PAGE under non-reduced and reduced conditions, variation in polypeptide pattern was analyzed for their comparative studies. The polypeptides, under non-reduced conditions, were ranged between the mol wt. 14.5- 108 kDa, while the polypeptides under reducing conditions were resolved in the range of mol wt. 8-98 kDa (Fig. S1a and S1b). Some of the polypeptides of mol wt. 108, 74.5, 74, 68, 48, and 40 kDa were observed in non-reducing gels but were reduced and disappeared in the reducing conditions. The new polypeptides of mol wt. 61, 55, 44.5, 41.5, 38.5, 38, 36, 35.0, 34, 33, 32.5, 31.5, 28, 13, 10.5, 9.5, 8.5 and 8.0 kDa were resolved under reducing conditions (Fig. S1b). The variation in polypeptide patterns among malt and feed barley varieties; among two rows and six rows barley lines; and salinity tolerant (ST) and salinity susceptible (SS) lines was observed as the molecular weight of protein bands were varying in different regions. Based on the banding profile of proteins gels were divided into five regions; zone A (73-98 kDa), zone B (53-68 kDa), zone C (37.5-51kDa), zone D (25.5-35 kDa) and zone E (8-18 kDa). The polypeptide pattern analysis in these regions revealed that region A had three polypeptide patterns A1, A2, and A3, region B had fourteen polypeptide patterns B1 to B14, region C had eighteen polypeptide patterns C1 to C18, region D had thirteen polypeptide patterns D1 to D13, and region E had two polypeptide patterns E1 and E2 (Table S1). When the polypeptides pattern of malt and feed barley were studied on the SDS-PAGE gel, the electrophoretic profiles of malt barley showed three different polypeptide patterns in region A (A, A2, and A3), eight different polypeptide patterns each in region B (B1, B2, B5, B7, B9, B12, B13, and B14) and C (C1, C2, C4, C5, C7, C8, C13, and C15), five different patterns in region D (D2, D5, D6, D8, and D10 ) and two polypeptide patterns in region E (E1 and E2). The polypeptide patterns of malt and feed barley in different regions have been summarized in Table S1. In region A, the pattern of mol wt. 98, 91.5, 82.5, 73 kDa (A1) occurred only in genotype K 551, the pattern of mol wt. 93, 82.5, 73 kDa (A2) occurs in five barley genotypes Alfa 93, BCU 73, DL 88, DWRB 73, and DWRUB 52. The polypeptide pattern of mol wt. 82.5, 73 kDa occurred in barley lines DWR 28, RD 2503, and RD 2668. The polypeptide pattern analysis of two rows barley exhibited two different polypeptide patterns in region A (A2 and A3), five different polypeptide patterns each in region B (B1, B2, B5, B13, and B14) and C (C1, C2, C7, C8, and C15). The polypeptide patterns of two rows and six rows barley in different regions has been summarized in Table S2. The polypeptide pattern analysis of salinity tolerant barley displayed three different polypeptide patterns, each in region A (A1, A2, and A3), region B (B3, B6, and B8), and region C (C6, C7, and C8), two different polypeptide patterns each in region D (D2 and D6) and E (E1 and E2). The polypeptide patterns of salinity tolerant and salinity susceptible barley in different regions has been summarized in Table S3.
Based on the electropherogram of total seed storage proteins of 34 different Indian barley lines, the polypeptide polymorphism percentage was calculated and analyzed among different barley lines with different characteristics (Fig. 1). The electrophoretic banding patterns of total seed protein of malt, feed, two-row, six-row, salinity tolerant, and salinity susceptible barley lines using SDS-PAGE demonstrated the differences among them (Table S4). The differences or polymorphism in the protein was based on the appearance of some bands in few varieties and the disappearance of the same bands in few varieties, whereas few bands equally appeared in all varieties of barley, i.e., monomorphic bands. SDS-PAGE analysis revealed that a total of 37 polypeptides bands of different molecular weight ranging between 8 kDa to 98 kDa together in all barley lines were present, out of which 54%, 59%, 35%, 59%, 38%, and 59% polypeptides bands were polymorphic in malt, feed, two-row, six-row, salinity tolerant and salinity susceptible barley lines respectively. However, the percentage of monomorphic bands in malt and feed barley were 43% and 41%, in two-row and six-row barley 49% and 41%, in salinity tolerant and susceptible barley 46% and 41% respectively. The polymorphic bands of 98 and 91.5 kDa of zone A appeared only in K 551, NB 1, NB 2, and Azad but were absent in two-row barley. In zone B, the polymorphic band of 65 kDa was present only in DL 88, RD 2552, and RD 2660 but absent in all two-row barley along with 54 kDa protein band, 54 kDa protein band was present in NB 2, RD 2052, RD 2503, and K 551. Another protein band of 55.5 kDa of this zone was absent in all salt-tolerant barley lines and 54 kDa protein, 55.5 kDa protein band was present only in Azad, k 560, HBL 316, DWRUB 52, DWRB 73. A polymorphic protein band of 51 kDa was absent in all salinity tolerant barley lines in zone C. In zone D, the polymorphic protein band of 28 kDa was absent in all-malt, six-row, and salinity tolerant barley lines but present in the feed, six-row, and salinity susceptible barley lines that include the PL 419, NB 2, RD 2592, and RD 2624. The protein band of 8.5 kDa was present in all barley lines, including NB 1, NB 3, RD 2552, RD 2668, K 551, K 560, K 603, BCU 73, and DWR 28.
Electrophoretic variations in the polypeptides of hordein fractions
SDS-PAGE analysis of the hordein fractions demonstrates variations between salt-tolerant and susceptible salt lines, between two-rows and six-rows, and between malt and feed barley lines. In this study, variations were observed in hordein polypeptides from seeds of cultivars studied. Arrays of 13 to 20 polypeptide bands were observed per genotype (Fig. 2). The band size of the hordein fraction was ranged from 31 to 96.5 kDa. Other studies of hordein fractions by Lee et al. (2010) reported the fraction ranges between 31-97.4 kDa, by Binott et al. (2017) ranged between 33-96 kDa). When SDS-PAGE gel were observed polypeptide bands in salt-tolerant line DL 88 (88.0 kDa, 83.5 kDa, 47.0 kDa, and 45.0 kDa), NDB 1173 (93.0 kDa, 88 kDa, 83.5 kDa, 47 kDa, 39 kDa, and 32 kDa), NB 3 (88 kDa, 83.5 kDa, 36.5 kDa, 47 kDa, and 45 kDa), NB 1 (88 kDa, 83.5 kDa, 36.5 kDa, 47 kDa, 45 kDa, and 31 kDa). Polymorphism in polypeptides patterns was observed in the region of C-hordein and D- hordeins. The salt-tolerant lines protein amount in B-hordein and γ- hordein was less than salt susceptible lines. Variations in B-hordein among barley lines were observed. Some lines share a similar pattern in B-hordein among groups but also a variation to other groups, B- hordeins of DWR 28, DWRUB 52, K 551, K 560, PL 419, PL 426, PL 751, and RD 2508 exhibit variations among B-hordeins of HBL 113, NB 1, and RD 2715 exhibit similar, BCU 73, HBL 316, RD 2552, and RD 2592 exhibits similar B- hordein pattern, K 508 and K 603 showed a similar pattern.
Phylogenetic relationship among 34 barley varieties
The dendrogram was constructed through cluster analysis which suggests a remarkable diversity among 34 Indian barley varieties. The varieties studied were grouped into seven non-overlapping clusters (Fig. S2). In cluster IV, a maximum number of varieties constituted 12 NDB 1173, PL 751, PL 426, DL 88, DWRB 73, RD 2035, HBL 316, RD 2668 DWRUB 52, RD 2508, JB 58, and K 560. Two varieties NDB 1173 and DL 88, were salinity tolerant. Four varieties DL 88, DWRB 73, DWRUB 52, and RD 2668 were malt varieties, while DWRB 73, DWRUB 52, and RD 2668 were two-row barley. Cluster III and V both consist of 6 varieties each. Cluster III comprises HBL 113, Alfa 93, K 603, VLB 56, PL 419, and DWR 28 barley varieties, and among these varieties, Alfa 93 and DWR 28 were two-row, malt barley variety, whereas HBL 113 was two-row and feed barley variety. Cluster V comprises VLB 85, BCU 73, BH 393, K 508, RD 2715, and RD 2592. BCU 73 was two-row and malt variety among these varieties, whereas RD 2715 was dual purpose barley. Cluster VI and VII were constituted by three varieties each. In cluster VI, barley varieties RD 2624, RD 2660, and RD 2552 were present, and RD 2552 was salinity tolerant barely. In cluster VII, barley variety NB 2, K 551, and Azad were present, and K 551 was six-row malt barley. Cluster I and II possessed only two varieties each, in cluster I, NB 1 and NB 3 were present, and both varieties were salinity tolerant six-row barley. In cluster II, barley varieties RD 2052 and RD 2503 were present, and RD 2503 was six-row malt barley.
2-D Diagonal gel electrophoresis of seed proteins
For the analysis of the polypeptide subunit composition, 2D-diagonal gel electrophoresis was carried out. Since proteins are linked with disulfide bonds, significant changes in the polypeptide patterns of seed proteins under non-reducing and reducing conditions on the 1D gel were observed. The polypeptides separated under the non-reduced condition in the first dimension followed by separation in the second dimension in reducing condition demonstrated that the polypeptides having the disulfide linkage were reduced and moved as spot off of the diagonal (Fig. 3). Cleavage of inter or intramolecular disulfide linkages of proteins by 2-mercaptoethanol resulted in altered electrophoretic mobility of polypeptides. The intra-molecular cleavage of disulfide linkage resulted in a conformational change in the single polypeptide, and their mobility may increase or decrease on SDS-PAGE. Different subunits of polypeptides are linked with disulfide bonds and when treated with 2-mercaptoethanol, these disulfide linkage breaks and protein subunits resolved as different spots in 2-D gels. The polypeptides which do not have di-sulfide linkages were resolved on the diagonal, and the proteins outside the diagonal contained thiols in close proximity to form intramolecular disulfide bonds.
Inter-molecular disulﬁde linked polypeptides in salt-tolerant and susceptible barley lines showed polymorphism when they were cleaved by 2-mercaptoethanol (Table 2). In salt-tolerant line NB 1, the polypeptide of mol wt. 108 kDa was cleaved into 40 kDa (B1) polypeptide subunit while it was absent in the other four salt tolerant lines. The polypeptide of 108 kDa was cleaved into 61+40, 38 kDa (F1+ F2, F3) in Alfa 93, in BH 393 and K 508, the polypeptide 108 was cleaved into 63+40 kDa (G1+ G2, and H1+ H2). The polypeptide of 68 kDa of salt-tolerant line NB 1 was cleaved into 38 kDa (B2) polypeptide subunit, while in salt susceptible lines Alfa 93 and K 551 (I1), it was cleaved into 38+27 kDa (F4+ F5), and 40 kDa (I1) respectively. The polypeptide of mol wt. 48 kDa was cleaved into 33 kDa in all salt-tolerant and susceptible lines except K 551.
The polypeptides which were resolved above the diagonal are the intramolecular disulfide-linked polypeptides (Table 3). In the genotype DL 88 polypeptides of mol wt. 37, 31, 29.5, 27.5, and 9.5 kDa represented intra-molecular disulfide linkage that resolved above the diagonal with higher mol wt. 42, 40, 32, 29, and 11 kDa, respectively. The polypeptide of mol wt. 40, 31.5, 29.5, 27.5, and 9.5 kDa were cleaved off into 44, 38.5, 32, 29, and 11 kDa polypeptides, respectively, in NB 1 barley line. In NB 3, the polypeptides of 32 and 29 kDa were resolved above the diagonal after the breakdown of intra linked disulfide linkage of polypeptides 29.5 and 27.5 kDa. The polypeptide of mol wt. 37, 31.5, 29.5, and 27.5 kDa were cleaved off into 42, 38.5, 32, and kDa polypeptides, respectively, in NDB 1173 barley line. In RD 2552, another salt-tolerant barley line, only 37 kDa polypeptide exhibited the intra linked disulfide linkage as it cleaved off into 42 kDa polypeptide. The polypeptide 27.5 kDa was common among all salt-tolerant barley lines except RD 2552. Alfa 93 and K 551 exhibited six polypeptides in salt susceptible barley lines with intra linked disulfide linkage, while K 508 and BH 393 exhibited two each, and NB 2 exhibited only one intra linked disulfide linkage (Table 3).