The Protein Disulfide Isomerase gene family in bread wheat (T. aestivum L.).

BACKGROUND: The Protein Disulfide Isomerase (PDI) gene family encodes several PDI and PDI-like proteins containing thioredoxin domains and controlling diversified metabolic functions, including disulfide bond formation and isomerisation during protein folding. Genomic, cDNA and promoter sequences of the three homologous wheat genes encoding the "typical" PDI had been cloned and characterized in a previous work. The purpose of present research was the cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species. RESULTS: Eight new non-homologous wheat genes were cloned and characterized. The nine PDI and PDI-like sequences of wheat were located in chromosome regions syntenic to those in rice and assigned to eight plant phylogenetic groups. The nine wheat genes differed in their sequences, genomic organization as well as in the domain composition and architecture of their deduced proteins; conversely each of them showed high structural conservation with genes from other plant species in the same phylogenetic group. The extensive quantitative RT-PCR analysis of the nine genes in a set of 23 wheat samples, including tissues and developmental stages, showed their constitutive, even though highly variable expression. CONCLUSIONS: The nine wheat genes showed high diversity, while the members of each phylogenetic group were highly conserved even between taxonomically distant plant species like the moss Physcomitrella patens. Although constitutively expressed the nine wheat genes were characterized by different expression profiles reflecting their different genomic organization, protein domain architecture and probably promoter sequences; the high conservation among species indicated the ancient origin and diversification of the still evolving gene family. The comprehensive structural and expression characterization of the complete set of PDI and PDI-like wheat genes represents a basis for the functional characterization of this gene family in the hexaploid context of bread wheat.

Identification and validation of reference genes for quantitative RT-PCR normalization in wheat.

BACKGROUND: Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. RESULTS: The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and alpha-tubulin. CONCLUSION: The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions. The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species.

Molecular and phylogenetic analysis of MADS-box genes of MIKC type and chromosome location of SEP-like genes in wheat (Triticum aestivum L.).

Transcription factors encoded by MIKC-type MADS-box genes control many important functions in plants, including flower development and morphogenesis. The cloning and characterization of 45 MIKC-type MADS-box full-length cDNA sequences of common wheat is reported in the present paper. Wheat EST databases were searched by known sequences of MIKC-type genes and primers were designed for cDNA cloning by RT-PCR. Full-length cDNAs were obtained by 5' and 3' RACE extension. Southern analysis showed that three copies of the MIKC sequences, corresponding to the three homoeologous genes, were present. This genome organization was further confirmed by aneuploid analysis of six SEP-like genes, each showing three copies located in different homoeologous chromosomes. Phylogenetic analysis included the wheat MIKC cDNAs into 11 of the 13 MIKC subclasses identified in plants and corresponding to most genes controlling the floral homeotic functions. The expression patterns of the cDNAs corresponding to different homeotic classes was analysed in 18 wheat tissues and floral organs by RT-PCR, real time RT-PCR and northern hybridisation. Potential functions of the genes corresponding to the cloned wheat cDNAs were predicted on the basis of sequence homology and comparable expression pattern with functionally characterized MADS-box genes from Arabidopsis and monocot species.

Gene expression induced by chronic ozone in the Mediterranean shrub Phillyrea latifolia: analysis by cDNA-AFLP.

Seedlings of Phillyrea latifolia L., a Mediterranean shrub, were exposed for 90 days to 110 nl l(-1) ozone (O(3)). Comparison of the cDNA-amplified fragment length polymorphism (cDNA-AFLP) patterns for treated and control plants allowed the identification and cloning of 88 differential sequences induced by O(3). The differential expression of 67 cloned sequences was further confirmed by RT-PCR. The functions of 36 cloned sequences, corresponding to seven of the twelve gene functional classes of Arabidopsis, were presumed on the basis of their homology with characterized gene sequences. Ozone induction of genes homologous to 24 of the clones has been reported in other plant species, whereas the induction of the 12 remaining sequences has not been observed before. Ozone activation of these newly identified genes could be a result of the chronic exposure to low O(3) concentration, because in most previous studies, acute treatments, involving high O(3) dosages, were applied. Possible roles of the cloned sequences in the response of P. latifolia to O(3) and other causes of oxidative stress are discussed.

Identification and characterization of gene sequences expressed in wheat spikelets at the heading stage.

Through differential analysis of transcripts (SDDM), 85 cDNA sequences specifically or preferentially expressed in wheat spikelets at heading time were identified and cloned; 54 of them had significant homology with genomic, cDNA and protein and 16 with EST sequences. Among these 54 clones, 44 matched genes with known functions, whereas 10 detected homology with putative genes encoding proteins whose functions have been deduced on the basis of bioinformatic comparisons. Seventeen clones corresponded to genes that had never been cloned in cereals, 5 were related to wheat genes with known functions, and the remaining 32 to genes cloned in other cereals. On the basis of their presumed functions, the 54 clones were assigned to seven groups. The first four of them contained 40 sequences likely involved in floral organ morphogenesis and gametogenesis, and precisely (i) sequences involved in the morphogenesis of floral organs; (ii) sequences expressed in pollen and/or anther tissues; (iii) sequences encoding transcription factors; (iv) sequences involved in signal perception and transduction (kinases and LRR proteins). The expression patterns of these 40 sequences have been studied by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of transcripts from different tissues and spike organs of wheat.

Extension of the Messapia x dicoccoides linkage map of Triticum turgidum (L.) Thell.

A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the high-density Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci.