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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Oral Communication Abstract

CLONING OF b1-TUBULIN AND MOB1-LIKE AS CANDIDATE GENES FOR 2N EGGS IN ALFALFA*

BARCACCIA G., MENEGHETTI S., LUCCHIN M.

Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Università di Padova, Agripolis, Via Romea 16, 35020 Legnaro, Padova, Italy

gianni.barcaccia@unipd.it

apomeiosis, DD, cDNA, AFLP, ESTs, tubulins, Mps-one-binder (Mob)

Mutants showing features of apomixis have been documented in alfalfa (Medicago sativa L.), a natural sexual reproducing species (reviewed by Barcaccia and Veronesi 1999, available at http://genes.alfalfa.ksu.edu/TAG). To date, genes expressed exclusively in megaspores or the embryo sac of apomictic ovules have yet to be isolated. Our research deals with the analysis of gene expression during megasporogenesis in apomeiotic mutants of diploid alfalfa characterized by 2n egg formation at high frequencies. A differential display of mRNAs that combines cDNA-AFLP markers and bulked-segregant analysis (BSA) was carried out with the aim of selecting ESTs and cloning and characterizing candidate genes for apomeiosis. The approach enabled us to select either mutant- or wild type-specific transcript-derived fragments and to detect transcriptional changes potentially related to 2n eggs. Sequence alignments of a subset of 40 polymorphic clones showed significant homologies to genes of known function. A b-tubulin cDNA, highly expressed in the wild types and poorly expressed in the apomeiotic mutants, and a Mob1-like cDNA, qualitatively polymorphic between pre- and post-meiotic stages, were selected as candidate genes for apomeiosis because of their roles in the cell cycle. A number of clone-specific primers were designed for performing both 5’ and 3’ rapid amplification of cDNA ends (RACE) to obtain the full-lengths. After assembling, the b-tubulin clone was shown to be 1544 nt in length and to contain an ORF of 1281 nt. On the basis of multiple alignments, this cDNA clone of alfalfa, named A-b1-Tub, shares between 93% and 95% amino acid sequence identity and up to 98% of similarity with the vast majority of b-tubulins of higher plants, both monocots and dicots. The final Mob1-like clone was 839 nt long. Comparisons with other Mob1-like sequences (e.g. Arabidopsis) suggest that the coding sequence of 582 nt lacks a region at the 3’ end corresponding to 22 aa. This cDNA clone of alfalfa, named A-Mob1-like, showed significant amino acid sequence homologies with genes from a variety of organisms, including plants, insects, animals, humans, yeast and fungi. Southern blot hybridization revealed that both clones belong to a multi-gene family with a minimum of three genomic DNA members each. Northern blot hybridization of total RNA samples and in situ hybridization of whole buds enabled the definition of their temporal and spatial expression patterns in reproductive organs. Experimental achievements towards the elucidation of apomeiotic megasporogenesis in alfalfa are presented and discussed. Because apomictic development may be initiated by the inhibition of meiosis in the megaspore mother cell, the elucidation of megasporogenesis alterations responsible for unreduced egg formation can be crucial for the understanding of apomixis.

*The nucleotide and amino acid sequence data of the isolated and characterized cDNA clones are availbale in the GenBank sequence database under the accession numbers AY037824 (Alfalfa mRNA for Mob1-like) and AY037825 (mRNA for b1-Tubulin).