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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

MICROSCALE ORGANIZATION OF REPETITIVE SEQUENCES IN THE NORWAY SPRUCE GENOME (PICEA ABIES L., KARST.)

CATTONARO F., JURMAN I., ZUCCOLO A.

Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Università degli Studi di Udine, Via delle Scienze 208, 33100 Udine, ITALY

federica.cattonaro@dpvta.uniud.it

Norway spruce, gymnosperms, genome, retrotransposons, BAC library

The Pinaceae are by far the most important gymnosperm family both economically and ecologically. We used Norway spruce as a model for the analysis of the Pinaceae genomic organization.

Our previous analyses demonstrated that its very large genome (1C=15x10⁹bp) is largely composed by interspersed highly repetitive sequences, mostly gypsy and copia retrotransposons.

In order to elucidate the organization and relative distribution of the repeats, sixteen probes, representative of different highly repetitive DNA families, were hybridized to high density arrays of 3456 spruce BAC clones (average inserts length 50 kb). 1190 of the BAC clones showed strong signals when hybridised with our probes; in particular 390 of them showed strong signals when hybridised with 2 or more different probes. Twenty clones (insert lengths ranging from 45 to 100 Kb), showing positive signals when hybridised with 3 or more different probes, were digested with EcoRI and HindIII restriction enzymes and used in Southern blot experiments in order to better investigate their relative arrangement at the microscale level.

The insert sizes of a random subset of 100 BAC clones will be determined with the aim of verifying whether BACs that did not hybridize to the highly repetitive sequences isolated so far coincide with those containing small inserts. To the contrary, a lack of such a correlation will indicate the existence of genomic regions devoid of the most abundant retroelement families.

Six clones (size range from 85 to 100 Kb) containing representatives of 3 to 6 different retrotransposon families and/or other not characterized repetitive sequences, were used to construct subclone libraries, randomly shearing the BAC clones by nebulization, to use in shotgun sequencing projects. The complete sequences of these genomic regions will allow us to understand the reciprocal organization of repeats in Norway spruce intergenic regions.