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 - 1.34

Cloning S-locus sequences related to sporophytic self-incompatibility in Cichorium intybus L.

G. Barcaccia, S. Varotto, M. Soattin, M. Lucchin, P. Parrini

Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Facoltà di Agraria,

Università di Padova, Agripolis, Via Romea 16, 35020 Legnaro, Padova, Italy

chicory, S-locus receptor kinase (SRK), S-locus glycoprotein (SLG), gene expression

In chicory (Cichorium intybus L.) inbreeding is hampered by a sporophytic incompatibility mechanism that prevents both selfing and crossing between plants showing identical phenotype at the multiallelic S-locus. Little information is available on the incompatibility system of this species and a deeper knowledge on the genetics of this reproductive barrier is needed for its exploitation in breeding programs. In chicory the incompatibility reaction occurs soon after pollination: trinucleate pollen grains cannot adhere (self-pollen) or germinate (allo-pollen) on the dry stigma. Cytological features indicate that the incompatibility reaction is partially similar to the pollen rejection observed in Brassica species, where the sporophytic incompatibility mechanism has been partly elucidated at molecular level. The products of two closely related genes, an S-locus receptor-like kinase (SRK) and an S-locus secreted glycoprotein (SLG) with multiple alleles organized in distinct haplotypes, cooperate to function as molecules for the recognition and rejection of the incompatible pollen on the stigmatic surface. SRK and SLG share a common domain called the S domain. A number of S domain-related sequences, unlinked to the S locus, have been found not only in Brassica, but also in several genomes of both dicots and monocots. In spite of the numerous attempts made for identifying S-gene products using RT-PCR and degenerated primers constructed on the mostly conserved regions of the S-gene products of Brassica, no related sequences were identified in chicory. The cDNA-AFLP method allowed us to obtain information on gene expression level and on its changes during the incompatibility reaction: although transcripts differentially expressed between self-compatible (SC) and self-incompatible (SI) plants of a C. intybus x C. endivia hybrid progeny were identified, no S-locus related sequences were found. Primers designed by Nasrallah et al. (1987, Nature 326:617-9) on the coding sequence of S-locus alleles of B. oleracea were used to screen by PCR different cDNA samples obtained through reverse transcription of total RNA isolated from SI and SC plants of C. intybus. Primer combination A1-forward and D1-reverse revealed a 1200 pb product that was cloned into a plasmid and sequenced. Homologies for the cDNA clone were searched in public databases to compare nucleotide and translated sequences. The sequence of chicory (AJ551426) showed a 99% similarity degree with an S-locus glycoprotein of B. oleracea (CAA34254), while the alignment with the SLG conserved domain scored a 8e-59 value. Moreover, sequencing analysis of genomic AP-PCR fragments obtained with the M13 universal primer revealed by chance a 430 nt long polymorphic amplicon of C. intybus partially homologous to an S-locus gene of Raphanus sativus (AY052578). Structural homologies were searched in protein databases to compare its deduced amino acid sequence with known proteins. The most significant amino acid sequences were used for pair-wise alignments to point out shared regions with respect to the translated sequence of chicory adopted as query. The highest similarity estimates were found with S-locus receptor-like kinases of Brassica. Semi-quantitative PCRs with internal primers using ds-cDNA templates isolated from SC and SI plants of a cross between genotypes contrasting for the incompatibility reaction (FB4 of C. intybus and SA12 of the self-compatible C. endivia) confirmed the differential expression of the putative SRK transcript. Research is in progress and 30-mer primers specific of the chicory SRK-like clone will be designed to obtain the full-length by performing both the 5' and 3' RACE. Both the SLG member and the SRK-related marker will be assigned to linkage groups of a functional map of C. intybus.