Comprehensive functional analyses of expressed sequence tags in common wheat (Triticum aestivum)

Abstract

About 1 million expressed sequence tag (EST) sequences comprising 125.3 Mb nucleotides were accreted from 51 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including abiotic stresses and pathogen challenges in common wheat (Triticum aestivum). Expressed sequence tags were assembled with stringent parameters after processing with inbuild scripts, resulting in 37,138 contigs and 215,199 singlets. In the assembled sequences, 10.6% presented no matches with existing sequences in public databases. Functional characterization of wheat unigenes by gene ontology annotation, mining transcription factors, full-length cDNA, and miRNA targeting sites were carried out. A bioinformatics strategy was developed to discover single-nucleotide polymorphisms (SNPs) within our large EST resource and reported the SNPs between and within (homoeologous) cultivars. Digital gene expression was performed to find the tissue-specific gene expression, and correspondence analysis was executed to identify common and specific gene expression by selecting four biotic stress-related libraries. The assembly and associated information cater a framework for future investigation in functional genomics.

Figure 1.

Analysis of singlet sequence length and their genotype-wise distribution. (A) Sequence length distribution of singlet. (B) Genotype-wise frequency (%) of singlet (AT, Atlas; SC, Scout; CC, Chancellor; TC, Thatcher; CR, Cranbrook; HB, Halberd; CS, Chinese Spring; KT, Kitakei1354; NR, Norin4; VV, Valuevskaya).

Figure 2.

Distribution of contig length and their EST member constitution. (A) Sequence length frequency of contigs. (B) Number of EST members in contigs.

Figure 3.

Sequence similarity of wheat contigs with rice genome. Based on the result, the contig was grouped in rice chromosome wise.

Figure 4.

Functional classification of contig sequences based on GO categorization. Sequences were evaluated for their predicted involvement in molecular function, biological process, and cellular component.

Figure 5.

miRNA target sequence analysis in wheat contigs. The database bars indicate the available miRNA in the database and the hit bars indicate the number of wheat genes having miRNA target sequence. tae, Triticum; sbi, Sorghum bicolor; osa, Oryza sativa; zma, Zea mays; ath, Arabidopsis thaliana; mtr, Medicago truncatula; ghr, Gossibium hirsutum; ptc, Populus trichocorpa; bna, Brassica napus; gma, Glycine max; pta, Pinus taeda; sly, Solanum lycopersicum; bra, Brassica rapa; bol, Brassica oleraceae; cre, Chlamydomonas reinhardtii.

Figure 6.

Correlated clustering of wheat cDNA libraries based on gene expression (AT, Atlas; SC, Scout; CC, Chancellor; TC, Thatcher; CR, Cranbrook; HB, Halberd; CS, Chinese Spring; KT, Kitakei1354; NR, Norin4; VV, Valuevskaya).

Figure 7.

Schematic diagram explaining the comprehensive EST analysis. The software used in the respective step was mentioned in parallel.