Analysis of expressed sequence tags from cDNA library of Fusarium culmorum infected barley (Hordeum vulgare L.) roots

Fusarium culmorum is one of the most common and globally important causal agent of root and crown rot diseases of cereals. These diseases cause grain yield loss and reduced grain quality in barley. In this study, we have analyzed an expressed sequence tag (EST) database derived from F. culmorum infected barley root tissues available at the National Center for Biotechnology Information (NCBI). The 2294 sequences were assembled into 1619 non-redundant sequences consisting of 359 contigs and 1260 singletons using the program CAP3. BLASTX analysis for these sequences was conducted in order to find similar sequences in all databases. Gene Ontology search, enzyme search, KEGG mapping and InterProScan search were done using Blast2GO 3.0.7 tool. By BLASTX analysis, 41.7%, 7.7%, 3.2% and 47.4% of ESTs were categorized as annotated, unannotated, not mapping and without blast hits, respectively. BLASTX analysis revealed that the majority of top hits were barley proteins (43.5%). Based on Gene Ontology classification, 38.3%, 31.3%, and 16% of ESTs were assigned to molecular function, biological process, and cellular component GO terms, respectively. Most abundant GO terms were as follows: 157 sequences were related to response to stress (biological process), 207 sequences were related to ion binding (molecular function), and 160 sequences were related to plastid (cellular component). Furthermore, based on KEGG mapping, 369 sequences could be assigned to 264 enzymes and 83 different KEGG pathways. According to Enzyme Commission (EC) distribution; 94 sequences were transferases (EC2) while 70 sequences were hydrolases (EC3).


Background:
One of the most common and globally important problems of cereal breeding is Fusarium root rot (FRR) and Fusarium rot (FCR) diseases [1-3].In the development of these diseases, a fungi complex consisting of Fusarium culmorum pseudograminearum (Syn: Fusarium graminearum), Microdochium nivale (Syn: Fusarium nivale), Fusarium avenaceum acuminatum, Bipolaris sorokiniana, Gaeumannomyces graminis other species plays a major role [4].The surveys carried out in Turkey showed that F. culmorum was the most abundant among these fungi [5].In order to reduce the use of pesticide in is one of the most common and globally important causal agent of root and crown rot diseases of cereals.These diseases cause grain yield loss and reduced grain quality in barley.In this study, we have analyzed an expressed sequence ta infected barley root tissues available at the National Center for Biotechnology Information (NCBI).The 2294 sequences were assembled into 1619 non-redundant sequences consisting of 359 contigs and 1260 P3.BLASTX analysis for these sequences was conducted in order to find similar sequences in all databases.Gene Ontology search, enzyme search, KEGG mapping and InterProScan search were done using Blast2GO 3.0.7 tool.and 47.4% of ESTs were categorized as annotated, unannotated, not mapping and without blast hits, respectively.BLASTX analysis revealed that the majority of top hits were barley proteins (43.5%).Based on Gene % of ESTs were assigned to molecular function, biological process, and cellular component GO terms, respectively.Most abundant GO terms were as follows: 157 sequences were related to response to stress (biological process), 207 sequences were related to ion binding (molecular function), and 160 sequences were related to plastid (cellular component).Furthermore, based on KEGG mapping, 369 sequences could be assigned to 264 enzymes and 83 different KEGG pathways.According to Enzyme Commission (EC) distribution; 94 sequences were transferases (EC2) while 70 sequences barley, ESTs, root rot and crown rot diseases, Fusarium culmorum One of the most common and globally important problems of Fusarium crown In the development of these diseases, a Fusarium culmorum, Fusarium Microdochium Fusarium avenaceum, Fusarium annomyces graminis and .The surveys carried out in was the most abundant .In order to reduce the use of pesticide in farming and struggling with the disease, it is cr investigate the mechanisms of natural resistance Investigation of resistance at the molecular level involves mapping of quantitative trait loci

Analysis of expressed sequence tags from cDNA infected barley
Vezneciler, Istanbul, Turkey; 34134 Vezneciler, Istanbul, is one of the most common and globally important causal agent of root and crown rot diseases of cereals.These diseases cause grain yield loss and reduced grain quality in barley.In this study, we have analyzed an expressed sequence tag infected barley root tissues available at the National Center for Biotechnology redundant sequences consisting of 359 contigs and 1260 P3.BLASTX analysis for these sequences was conducted in order to find similar sequences in all databases.Gene Ontology search, enzyme search, KEGG mapping and InterProScan search were done using Blast2GO 3.0.7 tool.and 47.4% of ESTs were categorized as annotated, unannotated, not mapping and without blast hits, respectively.BLASTX analysis revealed that the majority of top hits were barley proteins (43.5%).Based on Gene % of ESTs were assigned to molecular function, biological process, and cellular component GO terms, respectively.Most abundant GO terms were as follows: 157 sequences were related to response to stress on binding (molecular function), and 160 sequences were related to plastid (cellular component).Furthermore, based on KEGG mapping, 369 sequences could be assigned to 264 enzymes and 83 different tion; 94 sequences were transferases (EC2) while 70 sequences farming and struggling with the disease, it is crucial to investigate the mechanisms of natural resistance [6].
Investigation of resistance at the molecular level involves and identification of .On the other hand, induction of genes from pathways after pathogen infection strongly indicates their relation to the plant defense system

EST Processing
VecScreen tool (http: // www.ncbi.nlm.nih.gov/tools/vecscreen /) was used to find regions derived from vectors and vector contaminations were manually removed.The clean sequences were assembled into contigs and singletons with the CAP3 program [18].

Functional Annotation of ESTs
Functional annotations were performed in 3 steps by using Blast2GO 3.0.

Statistics of BLASTX results
In order to assign a putative function, Blast2GO According to GO Level distribution of ESTs, GO levels varied from 1 to 11 and total number of annotations were 4448 (Term Filter Mode: by Sequence Count, Term Filter Value:5).Based on the gene ontology category in biological process, 157 sequences were related to response to stress (GO: 0006950) while 76 sequences were associated with carbohydrate metabolism (GO:0005975), and 74 sequences were related to cellular amino acid metabolism (GO:0006520).In molecular function, 207 sequences were related to ion binding (GO: 0043167) and 70 sequences were related to oxidoreductase activity (GO: 0016491).With respect to cellular component ontology, the subcellular localization of 160 sequences was plastid (GO: 0009536), for 148 sequences was plasma membrane (GO: 0005886), and for 113 sequences was mitochondrion (GO: 0005739).A Multi-Level Pie Chart representation was used to display GO graphs (Figure 1).

Conclusion:
Root and crown rot disease is one of the most serious problems for cereal breeding.Despite this fact, very little information is available regarding the molecular mechanisms of such diseases.In this study, we performed EST analysis in order to investigate transcriptome during the early stages of colonization of barley roots by F. culmorum.After evaluation of 2294 ESTs in a F.culmorum-infected barley cDNA library, 157 response to stress-related genes were identified.264 enzymes were mapped to 83 different KEGG pathways.These results allow to identify a pool of stress and/or defense-related candidate genes.Primer design against those genes will allow to assess comparatively expression patterns of those genes in F. culmorum-resistant and F. culmorum-susceptible barley cultivars.

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resistance genes [8].On the other hand, induction of genes from several pathways after pathogen infection strongly indicates their relation to the plant defense system Expressed Sequence Tags (ESTs) database is a collection of short-single sequences of cDNA copies of mRNA that are expressed under different conditions.ESTs represent part of the Hypothesis © 2015 Biomedical Informatics

Figure 1 :
Figure 1: Gene Ontology annotation: Sequence distribution of Cellular component.

Table 1 (see supplementary material).
3.0.7 tool was used.All of the contigs and singletons were subjected to BLASTX analysis for homology search.Afterwards, mapping and annotation were performed.Data distribution of 1619 ESTs were as follows: 52 sequences with blast hits, 125 sequences with mapping and 674 sequences with GO-Slim annotation.768 sequences were without blast hits and removed because of lack of annotation.According to Top Hit species distribution of BLAST Top-Hits, the majority of top matches were H. vulgare KEGG pathways included cysteine and methionine metabolism (12 enzymes), amino sugar and nucleotide sugar metabolism (12 enzymes), and starch and sucrose metabolism (12 enzymes) and carbon fixation in photosynthetic organisms (7 enzymes).InterProScan tool was used to predict conserved domains in corresponding translated protein sequence.InterProScan of ESTs resulted in 1028 sequence of no InterPro, 591 sequences of InterPro and 223 sequences of GOs.