Defining the transcriptome assembly and its use for genome dynamics and transcriptome profiling studies in pigeonpea (Cajanus cajan L.)

Abstract

This study reports generation of large-scale genomic resources for pigeonpea, a so-called 'orphan crop species' of the semi-arid tropic regions. FLX/454 sequencing carried out on a normalized cDNA pool prepared from 31 tissues produced 494 353 short transcript reads (STRs). Cluster analysis of these STRs, together with 10 817 Sanger ESTs, resulted in a pigeonpea trancriptome assembly (CcTA) comprising of 127 754 tentative unique sequences (TUSs). Functional analysis of these TUSs highlights several active pathways and processes in the sampled tissues. Comparison of the CcTA with the soybean genome showed similarity to 10 857 and 16 367 soybean gene models (depending on alignment methods). Additionally, Illumina 1G sequencing was performed on Fusarium wilt (FW)- and sterility mosaic disease (SMD)-challenged root tissues of 10 resistant and susceptible genotypes. More than 160 million sequence tags were used to identify FW- and SMD-responsive genes. Sequence analysis of CcTA and the Illumina tags identified a large new set of markers for use in genetics and breeding, including 8137 simple sequence repeats, 12 141 single-nucleotide polymorphisms and 5845 intron-spanning regions. Genomic resources developed in this study should be useful for basic and applied research, not only for pigeonpea improvement but also for other related, agronomically important legumes.

Figure 1.

Distribution and read depth of sequence tags in the contigs. Number of 454 STRs aligning to form a contig ranged from 2 to >1000. A total of 36 152 contigs showed a read depth ranging from 2 to 5 tags, followed by 6755 contigs with read depth 6–10 tags, 5517 contigs with read depth 11–100 tags and 290 contigs with read depth ranging from 100 to 1000 tags. A maximum of 12 contigs showed a read depth of >1000 tags.

Figure 2.

Histogram plot of pair-wise synonymous distances of pigeonpea duplicated genes when compared with soybean. Histogram plot of percentage pair-wise distance to the synonymous distance value (Ks) showing a peak in pigeonpea at 0.06 which gives a divergence estimate of ∼4.9 Mya.

Figure 3.

Distribution of differentially expressed genes in SMD- and FW-responsive genotypes. Differential expression was calculated based on Log 2 value, with a threshold of less than −2 to greater than +2 number of differentially expressed gene was calculated for three SMD- and two FW-parental combinations. Comparison of expression values from susceptible parent to the resistant gave an estimate of up- and down-regulated genes in each cross.

Figure 4.

Hierarchical clustering of differentially expressed TAs within SMD- and FW-responsive genotypes. Hierarchial clustering of the gene involved in SMD- and FW-stress responses was done using HCE version 2.0 beta web tool. These two dendrograms illustrate the co-regulation of genes in resistant and susceptible genotypes under stress, (A) clustering of 5106 genes showing expression variation across six SMD-responsive genotypes (three resistant, ICPL 20096, ICPL 7035, BSMR 736, and three susceptible, ICPL 332, TTB 7, TAT 10). (B) Clustering of 3384 genes showing expression variation across four FW-responsive genotypes (two resistant, ICPL 87119, ICPL 99050, and two susceptible, ICPB 2049, ICPL 87091). Colour scale (from green to red) represents the range of expression level.