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

A NOVEL METHOD FOR DISTINCTNESS TESTING IN LOTUS

BARDINI M.*, MARIANI A.**, GIANÌ S.***, BREVIARIO D.***

* NIAB-Cambridge, UK

** IRMGPF-CNR, Perugia IT

Anna.Mariani@irmgpf.pg.cnr.it

*** IBV-CNR Milano IT

diego@ibv.mi.cnr.it

distinctness testing, genetic variability, DNA polymorphisms, PCR, tubulin

Innovative tubulin-based polymorphism DNA marker technique has been developed with the aim of establishing a novel fingerprinting platform for distinctness testing in Lotus. The genus Lotus contains many species both annual and perennial adapted to a wide range of ecological habitats. Some of them are of outstanding agricultural importance. However, Lotus species constitute complexes of closely related groups with very similar taxonomic characters, so that it is very difficult to distinguish among them. For instance there is still considerable argument over three taxa recognised as species or varieties, namely L. corniculatus s. str., L. tenuis Waldst and Kit, and L. alpinus Schleicher.

Considering the outstanding value of Lotus as a fodder crop but also the many problems related to its taxonomy, we found it necessary to reliably characterise the different species of Lotus to gain a better understanding of the germplasm structure and of the potentialities of the species and types it includes. Since morphological, chromosomal and even ecological characteristics have proved insufficient to identify the Lotus species we thought it necessary to use a molecular approach to the problem.

Accordingly, we sought to exploit some of known features of the genomic organisation of plant b-tubulin genes. More specifically we have relied, as the putative source for genomic DNA polymorphism, on the intron sequences of b-tubulin genes considered, in this respect, as special "intragenic" spacer regions. In fact, it was shown recently that intergenic spacer region (IGS) can be successfully used as the target sequences to check for genetic variability. We reason that intragenic spacer regions could work as well. Therefore, we set up a PCR-based protocol that can amplify b-tubulin intron regions supposed to be variable among the different b-tubulin isotypes that constitute the corresponding gene family in any given plant. Differences among taxa could then result from differences in the intron sequence or length of some or just one of these b-tubulin isotypes.

A preliminary protocol based on this assumption has been applied to the analysis of different varieties of Lotus. The results obtained with such an approach are quite promising although the pattern of amplification products that has been obtained is, for the moment, largely restricted to one band. Nevertheless the length of this band is different depending on the taxa from which the genomic DNA was originally extracted. This gives us confidence that this method could be further developed and may help in the characterisation of the phylogenetic relationship among the different types of Lotus (L. corniculatus L. alpinus L. tenuis and L. angustissimus) that have been analysed. More refined protocols of amplification and analysis of the amplified products obtained with this new tecnique are currently under investigation