Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 1.40

CHARACTERIZATION OF cDNA SEQUENCES DIFFERENTIALLY EXPRESSED IN WHEAT (TRITICUM AESTIVUM L.) SPIKELETS

M. CIAFFI, A.R. PAOLACCI, E. d’ALOISIO, O.A. TANZARELLA, E. PORCEDDU

Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo

ciaffi@unitus.it

wheat, flower development, heading stage, differential analysis, gene expression

The past decade has provided a wealth of information concerning the molecular and genetic control of flowering in higher plants. Numerous studies have led to the insight that inflorescence and flower development in higher eudicotyledonous species, such as the predominant model system Arabidopsis, are controlled by a hierarchy of interacting regulatory genes, most of them encoding transcription factors. Comparatively little is understood on genetic and molecular aspects of flowering in monocots. The elucidation of the molecular basis of grass flower development can be considered just at its beginning, since most of the genes involved in floral transition (flowering time genes), specification of floral meristem (meristem identity genes) and regulation of size and number of floral organs (floral meristem size genes and floral organ pattern genes) remain to be cloned and characterised. Moreover, most “floral organ identity genes” have yet to be detected in wheat, as only three MADS-box genes belonging to the AP1, AP3 and AG groups had been cloned. The aim of the present study was the isolation of cDNA sequences differentially expressed in wheat spikelets at the heading stage.

RNA was extracted from fully emerged spikes and flag leaves of Triticum aestivum cv. Chinese Spring. Purified mRNAs were analysed by a modified version of the Simple Differential Display Method (SDDM), which consists of the first-strand cDNA synthesis primed by random hexamers, followed by PCR amplification using random 10-mer oligonucleotides and then size separation of cDNA fragments by denaturing polyacrilamide gels. Out of 60 analyzed decamers, 38 detected 142 differential amplification products. Each differential band was extracted from the gel and amplified with the same primers used for the previous amplification. On the basis of northern hybridisation signals on RNA from spikelets and leaves, the 142 clones were assigned to four groups: group A included 40 clones which hybridised exclusively to the spikelet RNA; group B consisted of 25 clones whose hybridisation signals were higher in RNA from spikelets than in that from leaves; group C comprised 42 clones that showed similar hybridisation signals in both tissues; group D, finally, included 35 clones which did not detected any hybridisation signal in RNA from both spikelets and leaves. A total of 100 clones of the groups A, B and D were wholly sequenced. Group D clones were included because the lack of hybridisation signals in both analysed tissues could be due to very low expression level, which is peculiar of transcription factors and regulatory genes. Specific primers were designed for each of the 35 clones of group D and their expression in spikelets and leaves was checked by RT-PCR analysis. Differential expression in the spikelets was detected for 20 of the 35 clones of D group, whereas 8 clones were expressed in both spikelets and leaves and 7 primer combinations did not show any amplification product in both tissues. The search of nucleotide and deduced amino acid sequences of 85 clones, whose preferential expression in the spike had been previously shown either by Northern or RT-PCR analyses, in several databases allowed their assignment, on the basis of their putative functions, to four main functional groups: I) sequences involved in the morphogenesis of floral organs; II) sequences expressed in anther tissues and/or pollen; III) transcription factors; IV) kinases and LRR proteins. Some sequences probably related to the intense cell proliferation which takes places within the floral organs, such as cyclins, microtubule and centromeric proteins, or in the protein trafficking within the secretory pathway; were also cloned. The expression patterns of several cloned sequences have been studied by RT-PCR analysis of RNAs extracted from different plant tissues and floral organs of Triticum aestivum cv. Chinese Spring.