Enrico Porceddu

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.

PCR analysis of genes encoding allelic variants of high-molecular-weight glutenin subunits at the Glu-D1 locus.

Genes encoding high-molecular-weight (HMW) glutenin subunits, present in bread-wheat lines and cultivars, were studied by RFLP (restriction fragment length polymorphism) and PCR (polymerase chain reaction) analyses. In particular, allelic subunits of the x-or y-type, encoded at the Glu-D1 locus present on the long arm of chromosome 1D, were investigated. The variation in size, observed in different allelic subunits, is mainly due to variation in the length of the central repetitive domain, typical of these proteins. Deletions or duplications, probably caused by unequal crossingover, have given rise to the size heterogeneity currently observed. The possibility of using the PCR technique for a detailed analysis of HMW glutenin genes in order to obtain a more accurate estimation of the molecular weight of their encoded subunits, and the detection of unexpressed genes, is also described.

Development of a set of oligonucleotide primers specific for genes at the Glu-1 complex loci of wheat.

Specific amplification of the complete coding region of all six high-molecular-weight (HMW) glutenin genes present in hexaploid wheat was obtained by the polyerase chain reaction (PCR). Primers specific for the N-terminal region of the 1Dx gene and for the repetitive domain of the y-type HMW glutenin genes were also developed. Although the primers were constructed on the basis of the nucleotide sequences of HMW glutenin genes present in T. aestivum L. cv ‘Cheyenne’, they were very efficient in amplifying HMW glutenin genes of diploid and tetraploid wheat species. PCR analysis of HMW glutenin genes of T. urartu Tuman., T. longissimum (Schweinf. & Muschl.) Bowden and T. speltoides (Tausch) Gren. ex Richt, showed a high degree of length polymorphism, whereas a low degree of length variation was found in accessions of T. tauschii (Coss.) Schmal. Furthermore, using primers specific for the repetitive regions of HMW genes, we could demonstrate that the size variation observed was due to a different length of the central repetitive domain. The usefulness of the PCR-based approach to analyze the genetic polymorphism of HMW glutenin genes, to isolate new allelic variants, to estimate their molecular size and to verify the number of cysteine residues is discussed.

Storage-protein variation in wild emmer wheat (Triticum turgidum ssp.dicoccoides) from Jordan and Turkey. I. Electrophoretic characterization of genotypes.

Seed storage-protein variation at theGlu-A1,Glu-B1 andGli-B1/Glu-B3 loci in the tetraploid wild progenitor of wheat,T. dicoccoides, was studied electrophoretically in 315 individuals representing nine populations from Jordan and three from Turkey. A total of 44 different HMW-glutenin patterns were identified, resulting from the combination of 15 alleles in the A genome and 19 in the B genome. Twenty-seven new allelic variants, 12 at theGlu-A1 locus and 15 at theGlu-B1 locus, were identified by comparing the mobilities of their subunits to those previously found in bread and durum wheats. The novel variants include six alleles at theGlu-A1 locus showing both x and y subunits. The genes coding for the 1Bx and 1By subunits showed no or very little (3%) inactivity, the 1Ax gene showed a moderate degree (6.3%) of inactivity whereas the gene coding for lAy showed the highest degree of inactivity (84.8%). A high level of polymorphism was also present for the omega- and gamma-gliadins and LMW-glutenin subunits encoded by genes at the linkedGli-B1 andGlu-B3 loci (19 alleles). Some Jordanian accessions were found to contain omega-gliadin 35, gamma-gliadin 45, and LMW-2 also present in cultivated durum wheats and related to good gluten viscoelasticity. The newly-discovered alleles enhance the genetic variability available for improving the technological quality of wheats. Additionally some of them may facilitate basic research on the relationship between industrial properties and the number and functionality of HMW- and LMW-glutenin subunits.

Identification and molecular characterization of a large insertion within the repetitive domain of a high-molecular-weight glutenin subunit gene from hexaploid wheat

High-molecular-weight (HMW) glutenin subunits are a particular class of wheat endosperm proteins containing a large repetitive domain flanked by two short N- and C-terminal non-repetitive regions. Deletions and insertions within the central repetitive domain has been suggested to be mainly responsible for the length variations observed for this class of proteins. Nucleotide sequence comparison of a number of HMW glutenin genes allowed the identification of small insertions or deletions within the repetitive domain. However, only indirect evidence has been produced which suggests the occurrence of substantial insertions or deletions within this region when a large variation in molecular size is present between different HMW glutenin subunits. This paper represents the first report on the molecular characterization of an unusually large insertion within the repetitive domain of a functional HMW glutenin gene. This gene is located at the Glu-D1 locus of a hexaploid wheat genotype and contains an insertion of 561 base pairs that codes for 187 amino acids corresponding to the repetitive domain of a HMW glutenin subunit encoded at the same locus. The precise location of the insertion has been identified and the molecular processes underlying such mutational events are discussed.

Molecular characterization of a LMW-GS gene located on chromosome 1B and the development of primers specific for the Glu-B3 complex locus in durum wheat

Abstract Low-molecular-weight glutenin subunits (LMW-GS) represent a specific class of wheat storage proteins encoded at the Glu-3 loci. Particularly interesting are the LMW-GS encoded at the Glu-B3 locus because they have been shown to play an important role in determining the pasta-making properties of durum wheat. Genes encoding LMW-GS have been characterized but only a few of them have been assigned to specific loci. Notably, no complete LMW-GS gene encoded at the Glu-B3 locus has yet been described. The present paper reports the isolation and characterization of a lmw-gs gene located at the Glu-B3 locus. The clone involved, designated pLDNLMW1B, contains the entire coding region and 524 bp of the 5′ upstream region. A nucleotide comparison between the pLDNLMW1B clone and other LMW-GS genes showed the presence of some peculiar structural characteristics, such as short insertions in the promoter region, the presence of a cysteine codon in the repetitive domain, and a more regular structure of this region, which could be important for its tissue-specific expression and for the functional properties of the encoded protein, respectively.

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.

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.