**6. Association mapping between root traits and SSR markers**

In the present study, association mapping was applied for identification of association between root traits and SSR markers. Marker-trait association (MTA) based on polymorphism found in SSR markers applied on diverse wheat genotypes. Two different models were used for identification of QTLs associated with root traits as GLM (general linear model) and MLM (mixed linear model). GLM requires no kinship, and only Q matrix was used to determine association between markers and mean of phenotypic traits. The level of significance of P value was measured at *p* ≤ 0.01 in both GLM and MLM models. The QTLs having LOD values above 2.5 were considered for both GLM and MLM.

A sum of 102 molecular markers were used in the present study. Most of the markers showed high level of polymorphism. A total of 271 polymorphic alleles were generated. The alleles per locus ranged from 1 to 3 and an average of 2.63 per locus. Polymorphic information content (PIC) values of the markers were also calculated in the range of 0.03–0.59. Initially, in order to investigate the genetic diversity of the material, 100 wheat genotypes were grouped into different cluster populations (**Figure 1**). Population structure may lead to spurious association between marker and traits [38]. Therefore, a model-based approach was used for association mapping. Both the general linear model (GLM) and mixed linear model (MLM) were applied. The association analysis also concluded that hundreds of genotypes having different genetic backgrounds were classified into 13 distinct groups, viz., G1, G2, G3, G4, G5, G6, G7, G8, G9, G10, G11, G12 and G13.

**5. Root trait analysis and its significance to drought**

**Table 2.** List of PCR primers/molecular markers used in the current study.

To understand the performance of wheat crop under drought conditions, it is necessary to have a sound knowledge about root traits. Root traits vary from species to species on the base of water availability, growth, physiology and architecture [33]. Root surface area and root length in wheat crop play an important role in water uptake. A well-organized root system is necessary for efficient water uptake. In crops, fibrous root system consists of two types as seminal and nodal roots [34]. Well-developed root system could play positive role in water deficit (drought) areas. Root morphological traits greatly affect water and nutrient uptake. Herbaceous plants with fine roots, smaller diameter and greater root length are better adapted to dry conditions [35]. Root traits greatly influence the resource uptake and sustaining crop yield under drought stress conditions. For maximum grain yield in wheat, active and well-developed root system is necessary [36, 37].

**Marker Marker Marker Marker Marker Marker** *Cfd 15 Xbarc 154 Xwmc 232 Xgwm 372 Xwmc 177 Xgwm 293 Cfd 18 Xbarc 158 Xwmc 233 Xgwm 389 Xwmc 181 Xgwm 299 Xwmc 24 Xbarc 159 Xwmc 235 Xgwm 443 Xwmc 182 Xgwm 302 Xwmc 25 Xbarc 163 Xwmc 398 Xgwm 471 Xwmc 216 Xgwm 325 Xwmc 27 Xbarc 164 Xwmc 420 Xgwm 469 Xwmc 219 Xgwm 359*

*Xwmc 43 Xbarc 165 Xwmc 606 Xgwm 484 Xwmc 51 Xbarc 167 Xwmc 718 Xgwm 544 Xwmc 52 Xbarc 172 Xwmc 749 Xgwm 608 Xwmc 94 Xbarc 173 Xwmc 798 Xgwm 609 Xwmc 97 Xbarc 175 Xbarc 42 Xgwm 642 Xwmc 104 Xbarc 264 Xbarc 45 xgwm 908 Xwmc 147 Xgwm 4 Xbarc 76 Xgdm 3 Xwmc 149 Xgwm 10 Xbarc 101 Xgdm 5 Xwmc 153 Xgwm 33 Xbarc 127 Xgdm 6 Xwmc 154 Xgwm 37 Xbarc 128 Xgdm 19 Xwmc 157 Xgwm 55 Xbarc 134 Xgdm 28 Xwmc 161 Xgwm 60 Xbarc 137 Xgdm 33 Xwmc 163 Xgwm 71 Xbarc 140 Xgdm 46 Xwmc 166 Xgwm 99 Xbarc 141 Xgdm 114 Xwmc 167 Xgwm 111 Xbarc 144 VRN AF Xwmc 168 Xgwm 136 Xbarc 147 VRN B1 R3 Xwmc 169 Xgwm 194 Xbarc 148 PpD1 R1 Xwmc 175 Xgwm 261 Xbarc 149 PpD 1 R2*

44 Wheat Improvement, Management and Utilization

**Figure 1.** Population structure analysis of wheat genotypes based on SSR markers. (a) Graphical bar plot at k = 2 presenting two subgroups (G1 and G2). (b) Graphical bar plot at k = 13 presenting 13 subgroups (G1–G13). The X-axis shows accession numbers, and Y-axis shows subgroup membership.

#### **6.1. Total root length**

Total root length per unit ground area (La) is often considered to be directly related to the amount and rate of water uptake. Total root length (TRL) is associated with drought tolerance in wheat because it marks the spreading of roots in the soil and affects the resource uptake [39]. The genotype Pirsabak-85 ranked high on the base of TRL and R:S and considered to be the best for drought tolerance by extracting water stored in the deep soil layers. Further, in GLM model the SSR marker *Xgdm 5* on chromosome 2 was significantly associated with total root length, but no association of marker with TRL was found in MLM. The phenotypic variance (r2) was 0.10. The p value was recorded as 0.0016, and LOD is 2.78 as shown in **Figure 2**. The present research revealed that GLM model confirmed MTA for TRL was found to be located on chromosome 2D and the results are in accordance with previous results where MTA for TRL was reported on chromosome 2 at 3.4 cM [40].

For root fresh weight, the GLM model identified MTA associated with RFW, located on chromosome 5B. The marker *Xwmc 235* attributed to trace the QTL on specific chromosome for RFW. The phenotypic variance (r2) was found as 0.10, and LOD was 3.56 as shown in **Figure 3**. The previous report of Ayman, A.D., M. A.M. Atia, H.A. H. Ebtissam, A.H. Hashem and S.A. Sami. 2013. A multidisciplinary approach for dissecting QTL controlling high yield and drought tolerance-related traits in durum wheat. *Int. J. Agri. Sci. Res*.3: 99-116 confirmed that four QTLs are associated with RFW located on 2B, 5B, 6A and 6B chromosomes. Our results which did not localize other QTLs due to lesser number of markers have been used. Similarly, for root dry weight, the *PpD1* marker revealed marker trait association (MTA) for RDW in GLM model only. The MTA was found to be located on chromosome 2A having r2 0.41 and LOD of 2.7 as in **Figure 4**. These results were partly in agreement with results of [41] where the authors found that 5 QTLs for RDW were grouped in chromosomes 2A and 7A.

**Figure 2.** QTL identified for TRL on the basis of LOD in GLM.

**Figure 3.** QTL identified for RFW on the basis of LOD in GLM.

### **6.2. Maximum root length**

**6.1. Total root length**

46 Wheat Improvement, Management and Utilization

Total root length per unit ground area (La) is often considered to be directly related to the amount and rate of water uptake. Total root length (TRL) is associated with drought tolerance in wheat because it marks the spreading of roots in the soil and affects the resource uptake [39]. The genotype Pirsabak-85 ranked high on the base of TRL and R:S and considered to be the best for drought tolerance by extracting water stored in the deep soil layers. Further, in GLM model the SSR marker *Xgdm 5* on chromosome 2 was significantly associated with total root length, but no association of marker with TRL was found in MLM. The phenotypic variance (r2) was 0.10. The p value was recorded as 0.0016, and LOD is 2.78 as shown in **Figure 2**. The present research revealed that GLM model confirmed MTA for TRL was found to be located on chromosome 2D and the results are in accordance with previous

For root fresh weight, the GLM model identified MTA associated with RFW, located on chromosome 5B. The marker *Xwmc 235* attributed to trace the QTL on specific chromosome for RFW. The phenotypic variance (r2) was found as 0.10, and LOD was 3.56 as shown in **Figure 3**. The previous report of Ayman, A.D., M. A.M. Atia, H.A. H. Ebtissam, A.H. Hashem and S.A. Sami. 2013. A multidisciplinary approach for dissecting QTL controlling high yield and drought tolerance-related traits in durum wheat. *Int. J. Agri. Sci. Res*.3: 99-116 confirmed that four QTLs are associated with RFW located on 2B, 5B, 6A and 6B chromosomes. Our results which did not localize other QTLs due to lesser number of markers have been used. Similarly, for root dry weight, the *PpD1* marker revealed marker trait association (MTA) for RDW in GLM model only. The MTA was found to be located on chromosome 2A having r2 0.41 and LOD of 2.7 as in **Figure 4**. These results were partly in agreement with results of [41] where the authors found that 5 QTLs for RDW were grouped in chromosomes 2A and 7A.

results where MTA for TRL was reported on chromosome 2 at 3.4 cM [40].

**Figure 2.** QTL identified for TRL on the basis of LOD in GLM.

The maximum root length (MRL) evolved to capture deeper water from the soil under drought stress [42]. The Abdaghar-97 genotype recorded the maximum root length (MRL) to capture deep soil moisture in dry areas. Two MTAs were identified for MRL located on chromosomes 2A and 5B. MTA of chromosome 2A was marked by *Xgwm 10* having LOD (2.68) and that of 5B was

**Figure 4.** QTL identified for RDW on the basis of LOD in GLM.

attributed by *Xwmc 149* having LOD of 2.86 as in **Figure 5a** and **b**. So far only one QTL for maximum root length located on chromosome 4B has been reported [14]. Similarly, QTL was identified for MRL located on chromosome 5 at 158.5 cM [43]. Therefore, the MTA identified on 2A chromosome in the present study was not reported before and considered to be novel QTL for MRL.

#### **6.3. Number of nodal roots**

The bulk of roots would increase with the increase in number of tillers. Nitrogen uptake is affected by length and number of nodal roots [44]. The uptake of nutrients is 2–6 times

**Figure 5.** QTL identified for MRL on the basis of (a) GLM; (b) LOD in MLM.

more for nodal roots than seminal roots, and thus growing such genotypes in rain-fed areas would be desirable [45]. The results of the present study found Meraj-08 with high number of nodal roots and would be better for nitrogen and water uptake in rain-fed areas. As for as the number of nodal roots MTAs was concerned, the MTA for number of nodal roots located on chromosome 2B. SSR marker *Xwmc 175* recognized the MTA for NNR on chromosome 2B as shown in **Figure 6**. MTA for NNR was found at LOD 2.5, p value 0.00306, while the (r2) 0.17. Our results were accordance with result of [46] where the same QTL is reported on chromosome 2B. Two MTAs (QTLs) were found associated with root angle in GLM model. The MTAs were found to be located on chromosomes 7B and 6D. The MTA located on chromosome 7B recognized by *Xgwm 302* and that of 6D was identified by *Xwmc 749* as in **Figure 7***.* The results are consistent with previous results where QTL for RA was located on chromosome 7B at 86cM, and reported four QTLs for RA was located on chromosome 2A, 3D, 6A and 6D [47, 48].

#### **6.4. Root density**

attributed by *Xwmc 149* having LOD of 2.86 as in **Figure 5a** and **b**. So far only one QTL for maximum root length located on chromosome 4B has been reported [14]. Similarly, QTL was identified for MRL located on chromosome 5 at 158.5 cM [43]. Therefore, the MTA identified on 2A chromosome in the present study was not reported before and considered to be novel QTL for MRL.

The bulk of roots would increase with the increase in number of tillers. Nitrogen uptake is affected by length and number of nodal roots [44]. The uptake of nutrients is 2–6 times

**6.3. Number of nodal roots**

48 Wheat Improvement, Management and Utilization

**Figure 5.** QTL identified for MRL on the basis of (a) GLM; (b) LOD in MLM.

Root density (RDT) increases the efficiency of the root system and is considered to be the most important trait for uptake of phosphorus in wheat [42]. The genotype Soghat-90 ranked first on the base of RDT and is considered to be good for phosphorus uptake. Further, root density has been reported to be positively correlated with total root length, root diameter and water use efficiency [49]. Two MTAs were identified for root density (RDT) in both GLM and MLM models located on chromosomes 2B and 5B. The MTA for chromosome 2B was attributed by *Xwmc 175* and 5B by *Xwmc 235* having LOD of 3.28 and 2.5 as in

**Figure 6.** QTL identified for NNR on the basis of LOD in GLM.

**Figure 7.** QTL identified for RA on the basis of LOD in GLM.

**Figure 8a** and **b**. The results of the present study are in accordance with the earlier reports. Previously QTL for RDT has been reported on chromosome 2B at 158.5 cM and 5B at 47 cM [46, 50]. The number of seminal roots may result in better adaptation to drought conditions in wheat. Further, the number of seminal roots was negatively correlated with water use efficiency [51, 52]. The strong root system will reduce the WUE and hence will reduce biomass production. Therefore, it is needed to improve the root system function rather than a strong root growth for wheat survival in drought conditions. In the present study, the genotype Marwat-01 is recorded the highest NSR and is suggested to be good in more water uptake in rain-fed areas.

#### **6.5. Root diameter**

The high root diameter (RD) is associated with drought tolerance in wheat. The genotypes showing the highest RD are supported for drought stress tolerance due to large xylem vessels with increased resource uptake and are well organized in searching deep soil layers to extract water [53]. Further, total root length, maximum root length and root density increase or decrease extremely with a small change in root diameter and decrease in root diameter would increase crop yield under drought. Significant reduction in root diameter, total root length and root density under drought conditions has been previously documented [37, 54]. Two MTAs were identified for RD, each in GLM and MLM. Both MTAs were located in chromosome 5B, attributed by *Xwmc 233* having LOD 3.1 and 3.3 as in **Figure 9**. Our results were consistent with earlier reports, where QTLs for RD on chromosome 5B at 4.5 cM have been mentioned [55].

**Figure 8.** QTL identified for RDT on the basis of LOD in (a) GLM; (b) MLM.

**Figure 8a** and **b**. The results of the present study are in accordance with the earlier reports. Previously QTL for RDT has been reported on chromosome 2B at 158.5 cM and 5B at 47 cM [46, 50]. The number of seminal roots may result in better adaptation to drought conditions in wheat. Further, the number of seminal roots was negatively correlated with water use efficiency [51, 52]. The strong root system will reduce the WUE and hence will reduce biomass production. Therefore, it is needed to improve the root system function rather than a strong root growth for wheat survival in drought conditions. In the present study, the genotype Marwat-01 is recorded the highest NSR and is suggested to be good in more water uptake

The high root diameter (RD) is associated with drought tolerance in wheat. The genotypes showing the highest RD are supported for drought stress tolerance due to large xylem vessels with increased resource uptake and are well organized in searching deep soil layers to extract water [53]. Further, total root length, maximum root length and root density increase or decrease extremely with a small change in root diameter and decrease in root diameter would increase crop yield under drought. Significant reduction in root diameter, total root length and root density under drought conditions has been previously documented [37, 54]. Two MTAs were identified for RD, each in GLM and MLM. Both MTAs were located in chromosome 5B, attributed by *Xwmc 233* having LOD 3.1 and 3.3 as in **Figure 9**. Our results were consistent with earlier reports, where QTLs for RD on chromosome 5B at 4.5 cM have been

in rain-fed areas.

**Figure 7.** QTL identified for RA on the basis of LOD in GLM.

50 Wheat Improvement, Management and Utilization

**6.5. Root diameter**

mentioned [55].

**Figure 9.** QTL identified for RD on the basis of LOD in GLM.
