Pea Pisumsativumh GENE Sym35 Is Homologous To The Gene Nin Of Lotus Japonicus Regel

K. Larsen, A.Y. Borisov1, V.E. Tsyganov1, V.A. Voroshilova1, A.O. Batagov1,

L.H. Madsen2, Y. Umehara2, N. Sandal2, L. Schauser2, N. Ellis3, LA. Tikhonovich1,

J. Stougaard

'All-Russia Research Institute for Agricultural Microbiology, St.-Petersburg, Pushkin 8, Podbelsky chaussee, 3,168608, Russia

Aaarhus University, Laboratory of Gene Expression, Gustav Wieds Vej 10, DK-8000, Aarhus C, Denmark

3John Innes Centre, Norwich, NR4 7UH, UK

Despite the fact that garden pea (Pisum sativum L.) is an old and well-developed system for wide-range genetic and physiological research including studying plant-microbe interactions it is, unfortunately, not suitable for molecular genetics and gene manipulation due to large size of the genome in physical sense. A major advance in the study of pea symbiotic loci (to date more than 40) at the molecular level can therefore be achieved by taking advantage of other legumes more suitable for molecular genetics. One of those model legumes is Lotus japonicus (Regel.) K. Larsen. Recently the first genetically defined legume symbiotic gene, the Nin controlling early stages of nodule formation in L. japonicus, was cloned and sequenced. Using comparative phenotypic analysis the pea gene Sym35 was found as the most probable candidate to be a homologous gene for Nin. It opened up a possibility to clone the gene Sym35 using molecular probes developed for Nin. Clones containing different fragments of cDNA and genomic copies of the pea Nin homolog were identified in "Root Hair Enriched" and "Genomic" pea gene libraries kindly provided by Dr H. Franssen (Wageningen University, The Netherlands), respectively. Subsequent sequencing of the fragments gave information about primary structure of this gene in pea and showed high homology (44%). Based on the sequence information, the molecular markers (primers allowing specifically amplify the fragments of allelic variants of pea Nin in various lines) for pea gene homologous to the gene Nin of L. japonicus were developed. Those markers allowed establishing the fact that the genes Sym35 and pea Nin are in the same position on pea genetic map using co-segregation analysis and therefore they are the same gene. Detailed sequencing of three independent mutant alleles (lines SGENod"-l (sym35), SGENod"-3 (sym35) and RisNod8 (sym35)) and corresponding wild type ones (lines SGE and Finale) revealed specific mutations in all cases: early stop codons and substitution of amino acid, respectively.

Thus, the first pea symbiotic gene identified by means of chemical mutagenesis was cloned with the use of achievements of molecular genetics in model legumes that opens up a possibility for further comparative analysis of the gene Sym35!Nin function and regulation of expression in these two legume plant species.

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