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

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 3.41

SEARCH FOR FEMALE STERILITY GENES IN ALFALFA

BARONE P., ROSELLINI D., REALE L., VERONESI F.

Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali - Sezione Genetica e Miglioramento Genetico, Borgo XX Giugno 74, 06121 Perugia

roselli@unipg.it

callose, differential display, ovule sterility, Medicago sativa

The investigation of female sterility in plants is useful for two reasons, overcoming it for seed production improvement, and using it for manipulating the reproductive process.

The genetic control of a female sterility trait has been investigated in progenies of an alfalfa plant exhibiting 81% female sterility (B17) and a plant with low sterility (5%, P13). Sterility is associated with heavy callose (beta 1-3 and 1-4 glucan) deposition within the ovule, is sporophytic, shows continuous variation and 85% narrow-sense heritability. In a 50-plant B17xP13 F₁ population plants displaying 90-100% sterility were found. In some of these plants, sterility was accompanied by an arrest of pistil growth and, at flower maturity, the pistils were underdeveloped, partially necrotic, and contained aborted, callosized ovules. Cytological investigations have shown that in the fertile control, callose is present only during the meiotic process, in the walls of the megaspore mother cell and of the elements of the dyad and tetrad. On the contrary, in the sterile plants, callose is deposited before meiosis and before the pistil shows signs of developmental block. It invades the walls of the nucellus cells surrounding the sporogenous cells. This does not appear to prevent the formation of the megaspore mother cells; however, meiosis was never observed, but a condensation of chromatin indicating a meiosis I prophase stage was generally seen. Our investigations indicate a female-specific arrest of meiosis at prophase I or immediately earlier, accompanied by callose deposition in the nucellus. With the objective of finding the gene, or genes, responsible for sterility, we have excised flower buds from each of the 10 selected F1 plants described above. Five developmental stages were identified, spanning from the beginning of ovule differentiation to the flower maturity stage. Messenger-RNA differential display was carried out with the cDNA-AFLP technique. We have so far isolated, cloned and sequenced 65 polymorphic amplicons, the majority of which were absent in control leaf RNA amplification profiles.

From the comparison of the sequences of the isolated polymorphic bands (EST) with the nucleotide and protein of gene and Expressed Sequence Tag databases, some interesting homologies emerged:

- Glycine max beta-1,3 glucanase 7 (GI 2921322)

- Arabidopsis thaliana Cyclin 3b (GI 784946)

- Pisum sativum/A. thaliana beta-tubulin (GI 15239914)

- Medicago sativa Mitogen-activated protein kinase (GI 2499613).

The first EST, corresponding to an AFLP band expressed in the sterile plants only at the premeiotic and meiotic stages, is homologous to a soybean gene encoding a flower-specific, callose catabolism enzyme (Jin et al. 1999), that could be involved in callose deposition in the female sterile plants. The second sequence is homologous to an A. thaliana cyclin, and the corresponding AFLP band is absent in flower buds of sterile plants after the pre-meiotic stage. The alfalfa gene may be involved in the meiotic block of the megaspore mother cell. The third EST derives from an AFLP band present in fertile plants at the meiotic stage only, but present at all stages in sterile plants. The fourth EST derives from an AFLP band whose intensity decreases during flower development in sterile but not in fertile plants.