Medicago truncatula, an intergenomic vehicle for the map-based cloning of pea (Pisum sativum) genes : comparative structural genomic studies of the pea Sym2-Nod3 region

To determine the usefulness of M. truncatula as intergenomic vehicle for the positional cloning of pea genes it was studied whether these legumes are microsyntenic. These studies were focused on the pea Sym2 and Nod3 genomic regions. The M. truncatula orthologous genomic regions have been cloned and it was shown that these regions of the two legumes are microsyntenic. Both Sym2 and Nod3 play a key role in the pea- Rhizobium symbiosis, controlling Nod factor-structure dependent infection and autoregulation of nodule number, respectively.

A M. truncatula A17 BAC library was screened with a pea marker tightly linked to Sym2 and 11 clones were isolated. These clones formed three different contigs named C1, C2, and C3, which were extended to about twice their original size by chromosome walking resulting in contigs of 300 Kbp, 170 Kbp and 150 Kbp, respectively. Genetic and FISH mapping in M. truncatula revealed that the three contigs map on chromosome 5, and that C1 and C2 are tightly linked while C3 maps at a distance of 9 cM from C1/C2 on the same arm of this chromosome. By a combination of contig physical data and FISH it was estimated that C1 and C2 were separated by a gap of 30-40 Kbp. C1 and C2 were further linked to each other by screening an expanded version of the M. truncatula BAC library with the C1 and C2 contig-end subclones Mtg2511 and Mtg3556, respectively. Consequently, the small gap was closed by a 12 Kbp sequence linking C1/C2 which final size resulted in about 480 Kbp. Eight RFLP markers (including cDNAs and contig subclones) were isolated from C1/C2. Mapping of these markers using pea RILs and introgression lines demonstrated that C1/C2 represents the MedicagoSym2 -orthologous genomic region. Moreover, three markers showed recombinations between their pea homologous sequences and Sym2 , delimitating both the pea Sym2 region in the RILs and introgression lines, and the MedicagoSym2 -orthologous region . The MedicagoSym2 -orthologous region was delimitated to about 350 Kbp of C1/C2. In addition, by using a C2 subclone that encodes for a sequence highly homologous to the LRR-motif of the Cf4 and Cf9 tomato [ Licopersicon esculentum ] disease resistance proteins, a pea cDNA was isolated from a pea root hair library that also contains a LRR-domain highly homologous to that of Cf4 / Cf9 . The isolation of this pea RFLP marker demonstrates the use of M. truncatula as intergenomic positional-cloning vehicle of pea genes located within microsyntenic genomic regions. Furthermore, it was shown that the pea Sym2 -region is rich in Cf4 / Cf9 LRR-like sequences. The cloned C1/C2 Sym2 -orthologues candidates include receptor kinases and LRR-containing ( Cf4 / Cf9 -like, TMV -like) genes. Detailed analysis of 22 sequences from C1/C2 (including all RFLP markers) and the pea Sym2 -containing region showed that 4 of these sequences have homologues in c3, and that they are organized in clusters with a similar linear order in these contigs. This indicates that C1/C2 and C3 probably arose through duplication of a chromosomal segment.

By using the RNA differential display in combination with RILs and introgression lines, the tightly linked RFLP markers dd21.5 and Psc2.6 were isolated that are linked to the hypernodulating Nod3 locus and represent the closest markers mapping to the "south" of this locus on pea linkage group I in between Eil2 and Nod3 . These markers were used to identify the M. tuncatula A17 orthologous region with the aim to start the microsynteny-based positional cloning of Nod3 . The M. truncatula A17 BAC library was screened with these two markers. BAC clone 21F22 was isolated that contains the orthologue of dd21.5 . In addition 5 BAC clones were identified that co-hybridize with both dd21.5 and Psc2.6 , demonstrating the existence of M. truncatula genomic regions microsyntenic with the pea genomic region containing these two markers. BAC 21F22 was mapped by FISH on M. truncatula chromosome 4, revealing a local disruption of synteny at the dd21.5 locus between pea linkage group 1 and M. truncatula chromosome 5 that were syntenic for other markers (i.e. the markers isolated from the Sym2 -orthologous region, and the pea Eil2 marker). This finding reveals a chromosomal translocation that took place either in pea or in M. truncatula . However, it is unknown whether this translocation extends beyond the dd21.5 locus and includes the Nod3 locus. In addition to the strong FISH signal given by 21F22 on chromosome 4, weak signals were observed close to the telomeres of chromosome 5 and the position of one of these signals is comparable to the position of the C1/C2 Sym2 -orthologous region. Thus, it is possible that in spite of the translocation, chromosome 5 contains sequences with a low homology with those of the pea linkage group I segment containing dd21.5 . It remains to be determined whether the five microsyntenic BACs map in these chromosome 5 regions or whether they map in chromosome 4 and form a contig with BAC 21F22.

The data presented in this thesis set the basis for the microsynteny-based cloning of the Sym2 and Nod3 genes by using M.truncatula as intergenomic positional-cloning vehicle. In addition, the molecular resources generated in this thesis are useful to extend microsynteny studies at the Sym2 and Nod3 regions to other legume (e.g. Lotus japonicus ) and actinorhizal nodule-forming species, and also to non-nodulating related species from the Rosid I clade and non-nodulating less-related species (e.g. Arabidopsis and Brassica species that belong to the Rosid II clade). This will enable the identification of genes within these genomic regions that might be unique to nodule-forming plants engaged in symbiotic nitrogen fixation, representing a predisposition for nodulation at the ancestral root of the Rosid I clade. In other words, these studies will enable to answer one of the most interesting questions of plant biology and comparative genomics: whether the ability of legume and actinorhizal plants to establish a nodular symbioses, is given by unique properties that left their evolutionary "signatures" at the genome level.