Oronzo A. Tanzarella

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

BackgroundUsually 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.ResultsThe 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 α-tubulin.ConclusionThe 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.

Rapid and efficient detection of genetic polymorphism in wheat through amplification by polymerase chain reaction

The polymerase chain reaction (PCR) was used to amplify genomic DNA of several wheat genotypes. The oligonucleotides used as primers were the terminal sequences of a gamma-gliadin gene. The electrophoretic analysis of the PCR products showed specific bands which revealed both inter- and intra-specific genetic polymorphism among the examined genotypes. The technique is proposed as a very simple and efficient alternative to RFLP markers.

Introgression of Dasypyrum villosum chromatin into common wheat improves grain protein quality

Dasypyrum villosum (L.) Candargy (DV) is adiploid (2n = 14, VV genomes), allogamous grass of theMediterranean region. It may be hybridized with wheatand is thus a gene resource for wheat improvement. Westudied grain protein concentration andSDS-sedimentation (SED) as indicators of end-usequality. The latter is a good predictor of glutenstrength. A-PAGE and SDS-PAGE were used to identifymonomeric and polymeric seed storage proteins,respectively, to relate proteins of DV to those foundin Chinese Spring (CS), Triticum aestivum L.,wheat. Two full-sib lines of DV had high grain protein(19.3 and 20.3%), but one had very low mean SED (69mm) and one had very high (118 mm) based on onegreenhouse and one field test. CS had very low grainprotein (12.0%) and weak gluten (33 mm). Single-DVchromosome addition and substitution lines and twoDV-wheat recombinant lines all had higher grainprotein than CS (range 13.9 to 16.7%). SED valuesshowed a different pattern. CS=4V and CS=6V hadlow SED, 63 and 44 mm, similar to CS, whereas CS=1Vand full sib DV 200 had very strong gluten, 118 mm, asdid substitution lines CSċ1V (1A) and CSċ1V (1B), 125and 131 mm, respectively. One hybrid-derived line withDV-wheat 1V recombinant chromosome had SED of 99 mmand one line with a 6V added chromosome had SED of 64mm. The large positive effects of quality in the wheathaving DV chromosome 1V are believed to be due to DValleles at the Glu-V1 and Gli-V1/Glu-V3loci. DV chromosomes 4V and 6V did not contribute toimproved quality probably due to Gli-V2 and Gli-V3 which, as the orthologous loci in wheat, donot enhance wheat quality. Based on the positiveeffects of alleles on DV chromosome 1V in a breadwheat background, we conclude that D. villosumis a source of allelic diversity that can beconsidered for improving end-use quality in breadwheat.

Molecular aspects of flower development in grasses

The grass family (Poaceae) of the monocotyledons includes about 10,000 species and represents one of the most important taxa among angiosperms. Their flower morphology is remarkably different from those of other monocotyledons and higher eudicots. The peculiar floral structure of grasses is the floret, which contains carpels and stamens, like eudicots, but lacks petals and sepals. The reproductive organs are surrounded by two lodicules, which correspond to eudicot petals, and by a palea and lemma, whose correspondence to eudicot organs remains controversial. The molecular and genetic analysis of floral morphogenesis and organ specification, primarily performed in eudicot model species, led to the ABCDE model of flower development. Several genes required for floral development in grasses correspond to class A, B, C, D, and E genes of eudicots, but others appear to have unique and diversified functions. In this paper, we outline the present knowledge on the evolution and diversification of grass genes encoding MIKC-type MADS-box transcription factors, based on information derived from studies in rice, maize, and wheat. Moreover, we review recent advances in studying the genes involved in the control of flower development and the extent of structural and functional conservation of these genes between grasses and eudicots.

Phloem Cytochemical Modification and Gene Expression Following the Recovery of Apple Plants from Apple Proliferation Disease

Recovery of apple trees from apple proliferation was studied by combining ultrastructural, cytochemical, and gene expression analyses to possibly reveal changes linked to recovery-associated resistance. When compared with either healthy or visibly diseased plants, recovered apple trees showed abnormal callose and phloem-protein accumulation in their leaf phloem. Although cytochemical localization detected Ca(2+) ions in the phloem of all the three plant groups, Ca(2+) concentration was remarkably higher in the phloem cytosol of recovered trees. The expression patterns of five genes encoding callose synthase and of four genes encoding phloem proteins were analyzed by quantitative real-time reverse transcription-polymerase chain reaction. In comparison to both healthy and diseased plants, four of the above nine genes were remarkably up-regulated in recovered trees. As in infected apple trees, phytoplasma disappear from the crown during winter, but persist in the roots, and it is suggested that callose synthesis/deposition and phloem-protein plugging of the sieve tubes would form physical barriers preventing the recolonization of the crown during the following spring. Since callose deposition and phloem-protein aggregation are both Ca(2+)-dependent processes, the present results suggest that an inward flux of Ca(2+) across the phloem plasma membrane could act as a signal for activating defense reactions leading to recovery in phytoplasma-infected apple trees.

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.

Molecular characterization of gene sequences coding for protein disulfide isomerase (PDI) in durum wheat (Triticum turgidum ssp. durum).

The organisation of the durum wheat genomic sequence (3.5 kb) coding for protein disulfide isomerase (PDI), deduced by comparison between genomic fragments and cDNA sequences (1.5 kb) isolated from immature caryopses, is described. The gene structure consists of ten exons and nine introns. The presence of consensus sequences involved in splicing, such as intron-exon junctions and branchpoint, has been observed and discussed. Although the deduced wheat PDI amino acid sequence exhibited an overall identity of only 31% to that of human PDI, their modular architecture in terms of number, size, location and secondary structure-propensities of the constituent domains are remarkably similar. The comparison of the amino acid sequences with the eight available plant PDI-like sequences showed a high identity with four of them and low with the remaining ones. Analyses of transcription levels showed that the PDI mRNA was present in all analysed tissues, with much higher expression in immature caryopses.

Nucleotide sequence of a low-molecular-weight glutenin fromTriticum durum

The low-molecular-weight (LMW) glutenin subunits have been demonstrated to play a primary role in determining the viscoelastic properties of durum wheat gluten [ 1, 2]. In a previous paper we proposed PCR as a rapid alternative to isolate different allelic forms of wheat storage protein genes [3]. Specific genomic amplification of LMW genes from Triticum turgidum (L.) Thell. cony. durum (Desf.) MK. was obtained by PCR using as primers the first twenty nucleotides of the coding region, and twenty nucleotides of the complementary strand between position 927 and 946 of a LMW glutenin from T. aestivum [4]. The amplified products from T. durum cv. Lira were cloned and sequenced. The sequence of pLMW21 clone (856 bp long) was compared with the only LMW glutenin gene reported for T. durum (B.G. Cassidy and J. Dvorak, EMBL Data Library, accession number X51759) and with those from T. aestivum [4, 5, 6]; in all cases a general high degree of nucleotide homology was observed. Nucleotide comparison of pLMW21 clone with the glutenin gene from T. durum revealed presence of five insertions and three deletions and 46 nucleotide changes out of 845 nucleotides compared. Four insertions are represented by single triplets and one by two triplets, while the deletions are represented by triplets. The alignment of the nucleotide sequences of pLMW21 clone with the LMW gene from T. aestivum used as a model for primer construction [4] showed the presence of three deletions and two insertions, and 56 nucleotide changes out of 820 nucleotides compared. The three deletions have different extensions and encode a 14-peptide, a nonapeptide and a tetrapeptide, whereas the insertions code for an octopeptide and a tetrapeptide (Fig. 1). The comparison of nucleotide sequences showed that nucleotide substitutions, which cause amino acid changes, represent a high percentage of the total changes (50-70~o), but only few of them cause amino acid polarity change. Moreover, the inserted and deleted fragments are composed by a multiple number of triplets, demonstrating that the insertion and deletions containing incomplete triplets are negatively selected even in storage proteins, which are supposed to tolerate mutations more than other proteins. Finally, the nucleotide substitution at position 697 causes

RFLP analysis of Aegilops species belonging to the Sitopsis section

The phylogenetic relationships among theAegilops species belonging to the Sitopsis section were investigated using RFLP (restriction-fragment-length polymorphism) analysis. Twenty-five probes, each of which hybridised to oneor more restriction fragments located in the B-genomechromosomes of cultivated wheats, were used. At least one and in most cases two fragments were located in every B genome chromosome arm. Adendrogram derived from a cluster analysis of the complete RFLP dataset showed a subdivision of the species belonging to the Sitopsis section into one group comprising the species of the Truncata subsection and another group comprised of the species of theEmarginata subsection. Dendrograms also were produced using RFLP data from loci located in different combinations of only three chromosomes, and some of these showed different subdivisions of the species. This demonstrates the importance in obtaining reliable classification data of using probes that detect loci evenly distributed in the genome and located in each chromosomearm.

Cloning and sequencing of a PCR amplified gamma-gliadin gene from durum wheat (Triticum turgidum (L.) Thell. conv. durum (Desf.) MK.)

Abstract Specific amplification by PCR of gamma-gliadin genes from Triticum turgidum conv. durum cv. Langdon was obtained using high temperature for DNA-primers annealing. Three amplified bands were obtained and one of these was cloned and sequenced. Comparison of this nucleotide sequence with those of published gamma-gliadin and gamma-hordein genes revealed a high sequence homology. In particular, the comparison with pW1020 clone isolated from T. aestivum showed that the sequences derived from two genotypes differed by obly two nucleotides. The advantages offered by this approach for rapid gene isolation from different genotypes and its importance in molecular genetic analysis of wheat storage proteins are discussed.