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).     

Functional gene markers for polyphenol oxidase locus in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Higher polyphenol oxidase (PPO) activity in wheat kernels and flour has been implicated in the time dependent darkening of various end-products. Previous study conducted on a bread wheat (Triticum aestivum L.) doubled haploid (DH) mapping population derived from Chara (medium-high PPO) and WW2449 (low PPO) identified a major QTL for PPO activity located on the long arm of chromosome 2A. Physical mapping of SSR markers accounting for up to 84% of phenotypic variation for PPO activities suggests that the candidate PPO locus is localised in the deletion bin delimited by 2AL 0.77–0.85. In order to develop functional gene markers, nine wheat ESTs mapped to this deletion bin and partial PPO reference genes were explored for their sequence identities and linkage with PPO locus in a mapping population. In the present study, two markers: one SNP and one CAPS based upon BQ161439 sequence variation between the parents were identified which exhibited a tight linkage (0–0.6 cM) with the PPO loci designated as XTc1 and XPPO- _LDOPA. We also mapped the reference PPO gene (GenBank AY526268) characterised from developing kernels of wheat, on the long arm of chromosome 2A which exhibited a complete linkage with XPPO- _L DOPA locus. Results suggest that PPO variation displayed in the DH population from Chara/WW2449 is due to the same reference PPO gene. Allelic homoplasy of tightly linked markers, indicated that these markers are ‘diagnostic’ for the selection of low PPO gene in a range of germplasm being used in different Australian breeding programs. Identification and validation of ‘functional gene markers’ would facilitate in enhancing the selection efficiency for low PPO activity in wheat breeding programs.     

Functional integrated genetic linkage map based on EST-markers for a sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp.) commercial cross

The growing availability of ESTs provides a potentially valuable source of new DNA markers. The authors examined the SUCEST database and developed EST-derived markers. Thus to enhance the resolution of an existing linkage map and to identify putative functional polymorphic gene loci in a sugarcane commercial cross, 149 EST-SSRs and 10 EST-RFLPs were screened in the SP80-180 × SP80-4966 mapping population. With the markers already analyzed in the previous map, 2303 polymorphic markers were generated, of which 1669 (72.5%) were single-dose (SD) markers. Out of these 1669 SD markers, 664 (40%) were scattered onto 192 co-segregation groups (CGs) with a total estimated length of 6.261,1 cM. Using both genomic and EST-derived SSR and RFLP markers, 120 out of the 192 CGs were formed into fourteen putative homology groups (HGs). The EST-derived markers were subjected to BLASTX search in the SUCEST database, of which putative function was assigned to 113 EST-SSRs and six EST-RFLPs based on high nucleotide homology to previously studied genes. The integration of EST-derived markers improved the map, making it possible to consider additional fine mapping of the genome, and providing the means for developing ‘perfect markers’ associated with key QTL. To summarize, this paper deals with the construction of a genetic linkage map of sugarcane that is populated by functionally associated markers.     

Analysis of expressed sequence tags of flower buds in Lotus japonicus.

In order to study gene expression in a reproductive organ, we constructed a cDNA library of mature flower buds in Lotus japonicus, and characterized expressed sequence tags (ESTs) of 842 clones randomly selected. The EST sequences were clustered into 718 non-redundant groups. From BLAST and FASTA search analyses of both protein and DNA databases, 58.5% of the EST groups showed significant sequence similarities to known genes. Several genes encoding these EST clones were identified as pollen-specific genes, such as pectin methylesterase, ascorbate oxidase, and polygalacturonase, and as homologous genes involved in pollen-pistil interaction. Comparison of these EST sequences with those derived from the whole plant of L. japonicus, revealed that 64.8% of EST sequences from the flower buds were not found in EST sequences of the whole plant. Taken together, the EST data from flower buds generated in this study is useful in dissecting gene expression in floral organ of L. japonicus.     

Analysis of SSRs derived from grape ESTs

One hundred and twenty four microsatellites were isolated from analysis of 5000 Vitis expressed sequence tags (ESTs). A diversity of dinucleotide and trinucleotide simple sequence repeat (SSR) motifs were present. Primers were designed for 16 of these SSRs and they were tested on seven accessions. Ten of the sixteen primer pairs resulted in PCR products of the expected size. All ten functional primers were polymorphic across the accessions studied. Polymorphisms were evident at the level of cultivars, Vitis species, and between related genera. SSRs that were from the 3′ untranslated region (3′UTR) were most polymorphic at the cultivar level, the 5′ untranslated region (5′ UTR) SSRs were most polymorphic between cultivars and species, and those SSRs within coding sequence were most polymorphic between species and genera. These results show that EST-derived SSRs in Vitis are useful as they are polymorphic and highly transferable. With EST SSRs being applicable to studies at several taxonomic levels, the large number of SSRs (approximately 1000) that will be available from an expanded EST database of 45 000 will have many potential applications in mapping and identity research. Received: 4 June 1999 / Accepted: 21 September 1999     

Isolation and expression analysis of proline metabolism-related genes in Chrysanthemum lavandulifolium

Proline plays a significant role in plant resistance to abiotic stresses, and its level is determined by a combination of synthesis, catabolism and transport. The primary proteins involved are Δ¹-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PDH) and proline transporter (ProT). To utilise proline metabolism to improve the stress resistance of Chrysanthemum × morifolium, we isolated two P5CS-homologous genes (ClP5CS1 and ClP5CS2), one PDH gene (ClPDH) and four ProT-homologous genes (ClProT1-4) (GenBANK accession numbers: KF743136–KF743142) from Chrysanthemum lavandulifolium, which is closely related to chrysanthemums and exhibits strong resistance to stresses. Expression analysis of these genes in different organs and under various stresses indicated that ClP5CSs showed substantial constitutive expression, while ClPDH was only strongly expressed in the capitulum and was inhibited under most stresses. The expression patterns of four ClProT genes presented characteristics of organ specificity and disparity under stresses. Above all, the expression of ClProT2 was restricted to above-ground organs, especially strong in the capitulum and could be obviously induced by various stress conditions. Promoters of ClPDH and ClProTs contained many cis-acting regulatory elements involved in stress responses and plant growth and development. High levels of free proline were found in flower buds, the capitulum under the non-stress condition and later periods of stress conditions except cold treatment. Interestingly, organ specificity and disparity also exist in the level of free proline under different stress conditions. Our study indicates that ClProTs play significant roles in proline accumulation and stress responses, and that ClProT2 could be used to genetically modify the stress resistance of chrysanthemums. In addition, proline metabolism might be closely related to plant flowering and floral development.