PCA and Correlation Analysis of DEGs
To reduced data dimensionality and visualize genotype relationship under
stress and control conditions, we performed PCA analysis. The first
principal component (PC1) displayed 30.7% of total variation, while as
second principal component (PC2) explained 21.6% variation across the
data set (Fig. 3A). Scores plot between the PC1 and PC2 shows
clear separation of genotypes by PC1 under drought and control
conditions. However, based on the gene expression variation, wild-parent
(DTP/P1342618B) is clearly separated by PC2 from other three genotypes
viz., DSP, DTL & DSL; however, among these three genotypes tolerant
line (DTL) is more nearer to DTP/P1342618B genotype (Fig. 3A) .
This provides some explanation about the differences in the gene
expression pattern under drought and control conditions as well as in
the tolerant and sensitive genotypes.
To validate the results of PCA analysis, we performed correlation
analysis on the normalized expression values from all the samples, and
created a dendrogram (Fig. 3B). In roots, all four control
samples (DTP-C, DSP-C, DTL-C and DSL-C) were grouped in one cluster
(cluster-I), whereas the four stress samples (DTP-T, DSP-T, DTL-T and
DSL-T) were grouped in another cluster (cluster-II). In both clusters (I
& II), wild-tolerant parent was grouped alone in separate sub-cluster,
while other three genotypes are grouped together in another sub-cluster.
This can be explained by the fact as both introgression lines viz., DTL
and DSL are derived through repeated backcrossing of three times with
recurrent parent NN86-4/DSP, and after three repeated backcross 93.8%
of the recurrent parent genome is recovered in the introgression lines
(Collard et al. 2005), and hence due to this genome similarity both
introgression lines (DTL & DSL) are grouped together with recurrent
parent NN86-4/DSP in the same sub-cluster. Among these three genotypes
(DSP, DTL & DSL), the tolerant line DTL showed higher correlation with
wild-tolerant parent DTP in both Cluster I & II under both conditions;
however, under stress condition (Cluster II) correlation was relatively
higher compared to control conditions. This indicates that genes for
drought tolerance in DTL line are derived from DTP wild-parent. Hence,
the results of correlation analysis are in agreement with that of PCA
analysis (Fig. 3A & B ). These results revealed that DTP is
genetically more similar to DTL-tolerant line relative to DSL and DSP
genotypes.