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

IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN ISOGENIC MAIZE LINES AT A QTL FOR DAYS TO POLLEN SHED

GIULIANI S.*, DONNNISON I.**, SANGUINETI M.C.*

* Dipartimento di Scienze e Tecnologie Agroambientali, Università di Bologna, Italy

silgiul27@yahoo.com

** Institute of Grassland & Environmental Research, Aberystwyth, U.K.

maize, flowering time, isogenic lines, c-DNA AFLP, RDA

The transition from vegetative growth (the production of leaves) to flowering is the major developmental change occurring during the plant life cycle. Physiological studies have demonstrated that this transition is mainly localised in apical meristems which moves from the production of leaves and vegetative apex to the differentiation of reproductive structures.

A QTL for number of days to pollen shed (DPS) has been identified on maize chromosome 8L, bin 8.05, in different genetic background (Phillips et al., 1992. Proc. 47 Ann. Corn Sorghum Ind. Res. Conf., 135-150). A region of 6 cM on chromosome 8 has been shown to contain a QTL which accounts for nearly 80% of the phenotypic variation for DPS in a maize F2 population derived from the cross of two near-isogenic lines (NILs), namely N28 and N28EBC20. The line N28EBC20 (E = early) has been obtained after 20 backcrosses of the early line Gaspè Flint into N28. The allele for early flowering is able to induce an earlier transition of meristem to reproductive structure than the allele from N28 (Vladutu et al., 1999. Genetics, 153, 993-1007). This QTL is now the object of a positional cloning project in our laboratory.

An alternative way to identify genes responsible for the QTL governing DPS is the use of differential display techniques such as c-DNA Amplified Fragment Length Polymorphism (c-DNA AFLP, Bachem et al., 1996, Plant J. 9, 745-753) and Representational Difference Analysis (RDA, Lisitsyn et al. 1993, Science 259, 946-951). The use of NILs differing at a particular QTL region is to identify genes differentially expressed and related to the QTL. For this project we used N28 and C22-4 (N28EBC21) lines. Using the c-DNA AFLP and RDA techniques, trascriptional changes of the two cDNA pools have been monitored.

For each line, RNA was isolated from a pool of 200 apical meristems and from a pool of 50 leaves collected at stages comprised between the elongation of the first and the fifth leaves. For the cDNA AFLP technique, a modified protocol with only one (Mse) restriction enzyme was used. Thirty differentially expressed fragments were isolated from the gel, cloned in a plasmid (pGEM-T easy, Promega) and sequenced. The real differential expression of some of the fragments has been evaluated through the transfer of the DNA clones on a filter followed by the hybridization with DNA obtained from the first amplification of cDNA-AFLP technique. Two different experiments have been carried out, one with N28 and another one with C22-4 as probes. For the RDA technique the protocol described by Lisitsyn et al. (1993) has been modified for the use of cDNA. The same cDNA samples have been digested with DpnII and then enriched through two hibridizations followed by PCR. Of the 60 fragments which were cloned, a number has been sequenced. Sequencing of c-DNA AFLP and RDA fragments is in progress.