Field test

The experiment was carried out at the Campo Experimental Norman E. Borlaug (CENEB) located in the Yaqui Valley, Sonora (27° 22’ 10.2” N, 109° 55’ 51.5” W) during the autumn-winter cycle 2014- 2015. The trial consisted of nine lines of wheat flour (Table 1) and nine lines of durum wheat (Table 2) selected according to the susceptibility degree to karnal blunt manifested in three previous cycles. These were inoculated with T. indica sporidia during step boot stage or GS41-45 of ^{Zadoks et al. (1974)} scale. The inoculum production (isolation, propagation and sporidial concentration) was carried out using the methodology of ^{Fuentes-Dávila and Figueroa-López (2009)}.

Fuente: Programa de Mejoramiento Genético de Trigo (CENEB).

Table 1 Selected lines of bread wheat and its historical phenotypic response to T. indica 

Fuente: Programa de Mejoramiento Genético de Trigo (CENEB)

Table 2 Selected crystal wheat lines and their historical phenotypic response to T. indica. 

Evaluation of phenotypic response

To the crop, 30 spikes inoculated by variety were manually harvested and the percentage of infection was determined by the count of infected and healthy grains per spike per experimental unit and per line; the average percentage corresponds to the phenotypic response of each variety.

Oligonucleotides design

The sequences of the used oligonucleotides are listed in Table 3. Various programs were used for both editing and sequence alignment such as CLC Sequence Viewer and the NCBI Primer Designing Tool, adjusting the amplicon size to 200 bp. Part of the non-coding 3’ chain regions were included to ensure specificity between cystatine genes. Amplification of the oligonucleotides (sense and antisense) was tested by end-point RT-PCR and 2% agarose gel electrophoresis.

Table 3 Oligonucleotides for RT-PCR.  

Sampling

Spikes and foliar tissue were collected in duplicate for each line during three stages of phenological development, vegetative stage (SV), post inoculation (S1Pi), and post- anthesis (S2), corresponding to GS16-19, GS41-45 and GS69 respectively. These samples were immediately frozen with liquid nitrogen and stored at -80 °C until processing.

RNA extraction

Extraction of total RNA from 100-200 mg of frozen plant tissue was performed with Trizol^® (Invitrogen) reagent, following the manufacturer’s instructions with modifications. These consisted of the addition of a saline solution (Sodium Citrate 0.8 M + 1.2 M NaCl) during the precipitation phase in a 1:1 v/v ratio with isopropanol and during the purification phase with LiCl (8 M) per hour at 4 °C. The RNA was analyzed by electrophoresis to verify its integrity and its concentration and purity were determined by spectrophotometry in the NanoDrop 1000 (NanoDrop Products). Subsequently, the RNA was retrotranscribed to generate complementary DNA (cDNA) using the enzyme SuperScript (Invitrogen) according to the manufacturer’s instructions.

Real-time polymerase chain reaction (PCR)

Amplifications were performed using chemical detection with SYBR Green in reactions of 20 μl in 96-well plates in a C1000 thermocycler with CFX96 (Bio-Rad) real-time system.

Analysis of gene expression and correlation

The normalized expression of each gene was calculated using the comparative method of Ct (^{Livak and Schmittgen, 2001}). The abundance data of the transcripts were normalized with the average abundance of endogenous reference gene transcripts to obtain the normalized expression with the formula 2^-∆Ct, where: ΔCt= (Ct of the white gene - average Ct of the reference genes).

Pearson correlation analyzes were performed, considering percentage infection data against normalized expression values (2^-ΔCt) individually in leaf tissue and spikes. The Pearson correlation coefficient (R), the coefficient of determination (R²) and the level of significance at 95% confidence were obtained using the software package STATGRAPHIC Centurion XVI.

Percentage of infection

30 spikes per line were analyzed, classifying grains as “infected” and “healthy” according to the presence of typical damage by karnal bunt, then the percentage of spike infection and the average percentage per line were calculated. Table 4 shows that the wheat flour line BW3 showed the lowest percentage of infection (4.94%), while the line with the highest susceptibility was BW8 with 35.68%. In addition, the lines classified as intermediate response BW4, BW5 and BW6 were resistant (8.27%), susceptible (33.39%) and susceptible (32.94%) respectively.

Table 4 Phenotypic response of wheat to T. indica. 

Durum wheat has historically shown lower levels of infection compared to flour wheat. The percentages of karnal bunt infection in durum wheat are shown in Table 4, where the DW2 line shows a 0.19% infection. In contrast, the DW3 line was the most susceptible (60.33%). It is notorious the grouping of the varieties in 3 levels of resistance to T. indica, resistant (DW2, DW4, DW5 and DW9), susceptible (DW1, DW3 and DW7) and intermediate, which tend to be classified as resistant (DW6) and susceptible (DW8). It is possible to attribute phenotypic variations to genetic interactions in wheat lines, these variations may suggest the partial dominance of the partial carbon resistance attribute.

Standard expression (ΔCt)

For the calculation of gene expression it is required to normalize using expression data from one or more endogenous genes. These were selected from the literature based on homogeneity at different stages of development and tissues; EF1A standing out in various tissues under biotic stress conditions and TaWIN1 by its stability in flag leaves until the formation of milky grain (^{Tenea et al., 2011}).

The WC1, WC3 and WC5 transcripts were differentially expressed in resistant and susceptible genotypes at all development stages, and the normalized expression patterns were different in leaf tissue and spikes. This was determined using 95% Tukey multiple rank tests.

In leaf tissue, cystatin was differentially expressed during plant development (Figure 1). Expression levels of WC1 were significantly (p< 0.05) higher in the resistant lines BW3 in SV (1.015) and BW2 in S1 (1.61) and S2 (1.132). This pattern is repeated in the WC3 expression, with BW3 in SV (1.182) and BW2 in S1 (2.88) and S2 (2.186). On the other hand, the behavior of the WC5 levels was mixed, since it is observed that the greater expression in SV corresponds to the lines BW1 (0.003), BW3 (0.0026) and BW2 (0.002), the first one having a susceptible response value (16.31%). On the other hand, in the S1 the BW8 line was the one that most expressed WC5 (0.007), being this one the one that showed greater susceptibility (35.68%), whereas in S2 the lines BW1 and BW2 showed the highest values, 0.0043 and 0.0036 respectively.

Figure 1 Relative expression of cystatins WC1, WC3 and WC5 in wheat flour leaves during stages SV, S1 and S2. Literals indicate significant difference (p< 0.05).  

The spike constitutes a focal point of activity in wheat, the nutrients directed to the formation of the grain are mobilized and regulated towards here, is also where T. indica finds appropriate conditions to proliferate or reproducing; so that the level of expression of the cystatin can be affected by both the phenological stage and the presence of the pathogen. As shown in Figure 2, the normalized expression of cystatin was comparatively lower than that shown in leaf tissue (Figure 1) for WC1 and WC3, while WC5 was higher.

Figure 2 Relative expression of cystatins WC1, WC3 and WC5 in wheat flour leaves during S1 and S2 stages. Literals indicate significant difference (p< 0.05).  

The WC1 gene was more expressed in the BW5 susceptible line with 0.33 during S1, also during S2 the highest values were in the susceptible BW7 (1.222) and BW8 (0.568). In the expression of WC3 it is observed that during S1 the susceptible line BW5 followed by the resistant BW3 and BW9 showed the highest values (0.434, 0.362 and 0.346 respectively). The significant increase in the normalized expression values in S2 (post-anthesis stage) implies an inherent gene activity to the plant development in all evaluated varieties. However, since the normalized expression values of WC3 are greater than those observed for WC1 and WC5, it could suggest that the presence of the pathogen is affecting the expression of WC3, probably as part of a defensive system in the plant. The expression of WC5 decreases from S1 to S2 in most of the lines except for the resistant BW4 (0.583), the susceptible BW7 (0.324) and the resistant BW3 (0.163). When comparing these values with the controls, it was observed that in the controls the normalized expression of WC5 is higher, so that WC5 levels decrease in the presence of the pathogen.

In durum wheat (Figure 3), the resistant lines DW2, DW4, DW6 and DW9 showed significantly higher expression levels in SV and S1 for WC1, WC3 and WC5. During S2 stage the expression levels of WC1 and WC3 (including controls) decreased, so that in durum wheat the levels of WC1 and WC3 appear to be more affected by phenological development than by the interaction with the fungus. The expression patterns of WC5 varied little from S1 to S2; standing out the DW4 and DW6 resistant lines in S1, as well as DW8 and DW5 in S2. The above could indicate a background transcript of WC5 lower or similar to that of the endogenous genes.

Figure 3 Relative expression of cystatins WC1, WC3 and WC5 in durum wheat leaves during stages SV, S1 and S2.Literals indicate significant difference (p< 0.05).  

The expression patterns of cystatin in spikes of durum wheat (Figure 4) differ markedly from that shown in foliar tissue; WC1 as WC3 reached significantly lower levels. WC5 expression in spikes exceeded the levels reached in leaf tissue during S2, this behavior was also observed in flour wheats. The DW5 and DW9 lines excelled in the two growth stages in all three genes. The latter are resistant (2.45% and 1.24%).

Figure 4 Relative expression of cystatins WC1, WC3 and WC5 in durum wheat spikes during stages S1 and S2. Literals indicate significant difference (p< 0.05).  

In both, bread wheat and durum wheat the intense reaction produced during S2 was remarkable, this is in agreement with numerous investigations that have reported the occurrence of expression peaks of cystatin during the post-fertilization stage; also this expression is significantly stronger in resistant genotypes than in the susceptible ones (^{Gupta et al., 2010}; ^{Purwar et al., 2010}). These results indicate that increased expression of WC1, WC3 (in spikes) and WC5 (leaves) could be related to resistance due to the increase in S2, in the stage of greatest vulnerability to infection of T. indica, since the grain where the pathogen will colonize is formed.

Correlation analysis

The post-inoculation embedding step (S1) was considered focal point of cystatin gene activity; this behavior was correlated with the resistance level to karnar bunt in bread wheat spikes with the WC3 gene. This correlation follows the linear model with a coefficient of -0.709, a coefficient of determination of 50.30% and an equation of the line of WC3= 0.357966 - 0.0056264*(%) infection at 95% significance. The above allows to attribute 50.30% of the variation of infection percentage to the gene expression; that is, the increase in the relative expression of WC3 causes a decrease in the percentage of infection.

In the post anthesis stage, the behavior of the relative expression of WC5 in bread wheat leaves showed significantly correlation (p< 0.05) with the infection percentage, it was adjusted to the linear model with a line equation as follows: % Infection= 35.9286 - 4937.86*WC5. The correlation coefficient indicates that there is a moderately strong inverse relationship (-0.789) and a significant coefficient of determination (62.34%).

These results evidenced a significant relationship between the expression of cystatin and the karnal bunt resistance attribute; it also suggests some synergy between WC1, WC3 and WC5 by regulating the activity of cysteine proteases during seed maturation (^{Purwar et al., 2012}), when proteins must be accumulated for storage in response to the infection of T. indicates.