Generation and Analysis of a Large-Scale Expressed Sequence Tag Database from a Full-Length Enriched cDNA Library of Developing Leaves of <i>Gossypium hirsutum</i> L

<div><p>Background</p><p>Cotton (<i>Gossypium hirsutum</i> L.) is one of the world’s most economically-important crops. However, its entire genome has not been sequenced, and limited resources are available in GenBank for understanding the molecular mechanisms underlying leaf development and senescence.</p><p>Methodology/Principal Findings</p><p>In this study, 9,874 high-quality ESTs were generated from a normalized, full-length cDNA library derived from pooled RNA isolated from throughout leaf development during the plant blooming stage. After clustering and assembly of these ESTs, 5,191 unique sequences, representative 1,652 contigs and 3,539 singletons, were obtained. The average unique sequence length was 682 bp. Annotation of these unique sequences revealed that 84.4% showed significant homology to sequences in the NCBI non-redundant protein database, and 57.3% had significant hits to known proteins in the Swiss-Prot database. Comparative analysis indicated that our library added 2,400 ESTs and 991 unique sequences to those known for cotton. The unigenes were functionally characterized by gene ontology annotation. We identified 1,339 and 200 unigenes as potential leaf senescence-related genes and transcription factors, respectively. Moreover, nine genes related to leaf senescence and eleven MYB transcription factors were randomly selected for quantitative real-time PCR (qRT-PCR), which revealed that these genes were regulated differentially during senescence. The qRT-PCR for three <i>GhYLSs</i> revealed that these genes express express preferentially in senescent leaves.</p><p>Conclusions/Significance</p><p>These EST resources will provide valuable sequence information for gene expression profiling analyses and functional genomics studies to elucidate their roles, as well as for studying the mechanisms of leaf development and senescence in cotton and discovering candidate genes related to important agronomic traits of cotton. These data will also facilitate future whole-genome sequence assembly and annotation in <i>G. hirsutum</i> and comparative genomics among <i>Gossypium</i> species.</p></div

Analysis of GhYLS9 relationships.

<p>(a) Multiple sequence alignment of GhYLS9 and other homologous proteins in plants: <i>Arabidopsis thaliana</i> YLS9 (AB047812), <i>Casuarina glauca</i> HIN1 (ABZ80409), <i>Nicotiana tabacum</i> HIN1 (BAD22533), <i>Ricinus communis</i> SYP(XP_002532540), <i>Cucumis sativus</i> SYP24 (XP_004136508) and <i>Glycine max</i> SYP24 (XP_003554459). (b) Phylogenetic tree of these plant proteins constructed with MEGA 4 (c) Changes in transcript levels of GhYLS9 genes at each leaf development stage.</p

Frequency and distribution of ESTs among assembled contigs.

<p>Frequency and distribution of ESTs among assembled contigs.</p

The most abundant ESTs detected in the <i>Gossypium hirsutum</i> leaf library.

<p>The most abundant ESTs detected in the <i>Gossypium hirsutum</i> leaf library.</p

Sequence length distribution of upland cotton ESTs after assembly.

<p>Sequence length distribution of upland cotton ESTs after assembly.</p

The most abundant putative transcriptional factors (TFs).

a<p>Percent = (total number of unigenes)/(total number of putative TFs). There were 200 putative TFs.</p

Expression patterns of nine putative leaf senescence related genes from upland cotton.

<p>(a) Chlorophyll contents per fresh weight of leaves at each of four developmental stages. (b) Changes in transcript levels of the nine putative leaf senescence-related genes at each leaf developmental stage.</p

Analysis of GhYLS8 relationships.

<p>(a) Multiple sequence alignment of GhYLS8 and other homologous proteins in plants: <i>Arabidopsis thaliana</i> YLS8 (AB047811), <i>Hevea brasiliensis</i> YLS8 (XP_004148041), <i>Cucumis sativus</i> TRX4A (XP_004163626), <i>Medicago truncatula</i> TRX4A (XP_003590204), <i>A. thaliana</i> TRXU5(AED91278) and <i>Vitis vinifera</i> TRX4A (XP_002310072). (b)Phylogenetic tree of these plant proteins constructed with MEGA 4 (c) Changes in the transcript levels of GhYLS8 genes at each leaf development stage.</p

Phylogeny analysis of putative MYB transcription factors.

<p>Twenty-two putative cotton MYB transcription factors and thirty-one putative MYB transcription factors from other plant species were aligned and analyzed by neighbor-joining in MEGA4.</p

Comparison of the <i>Gossypium hirsutum</i> leaf EST library with those of other species.

<p>Comparison of the <i>Gossypium hirsutum</i> leaf EST library with those of other species.</p