ISSN 1022-7954, Russian Journal of Genetics, 2007, Vol. 43, No. 4, pp. 381–386. © Pleiades Publishing, Inc., 2007. Original Russian Text © T.V. Danilova, M.Yu. Kuklev, G.N. Andreeva, V.S. Shevelukha, G.I. Karlov, 2007, published in Genetika, 2007, Vol. 43, No. 4, pp. 482–488. 381 INTRODUCTION Wild silverleaf sunflower, Helianthus agrophyllus, is resistant to a number of diseases and parasites that affect cultivated sunflower, and is used in breeding pro- grams as a source of the resistance to Phomopsis stem canker, Diaporthe helianthi [1, 2], white mold, Sclero- tinia sclerotiorum [3], downy mildew, Plasmopara hal- stedii [4], sunflower broomrape, Orobanche cumana [5], and drought [6]. However, breeding of resistant forms of sunflower is restrained by the lack of effective methods of selection for resistance. In recent years, molecular marking of economically important traits of cultivated plants, including the resistance to different pathogens, has widely used. At present, resistance genes have been studied in many plants [7]. The resistance gene products (R-pro- teins) of monocotyledonous and dicotyledonous plants have conservative domains, the structures of which are similar, albeit the differences in the mechanisms of infection with pathogens and parasites. Depending on the presence of certain conservative regions, different classes of resistance genes are recognized. The proteins containing N-terminal nucleotide binding site (NBS) and C-terminal leucine-rich repeats (LRR), represent the products of the largest in number class of the resis- tance genes studied so far (NBS–LRR). The NBS–LRR proteins in turn are subdivided into subclasses in accor- dance with the N terminus structure. One of the sub- classes includes the proteins with TIR domains, homol- ogous to Toll receptor protein of Drosophila and human interleukin-1 receptor (TIR NBS–LRR). These proteins were found only in dicotyledons. They include the products of the N gene of tobacco, L6 gene of flax, and RPP5 gene of Arabidopsis. The second subclass com- prises the proteins without TIR domain (NBS–LRR). These proteins were described both in monocotyledons and dicotyledons, including, for example, the products of the Rx gene of potato and the Mlo gene of barley. Proteins of the third subclass are characterized by the presence of a leucine zipper structure in N-terminal region (LZ) (LZ–NBS–LRR). These are the products of the RPS2 gene of Arabidopsis, and Mi and Prf genes of tomato [8, 9]. Based on conserved amino acid sequences of NBS and LRR domains, degenerate primers were designed. PCR with these primers produced DNA sequences of putative resistance genes in many plant species. These sequences are called resistance-gene analogs (RGA) or resistance candidate genes. Genetic analysis of RGA showed that these sequences were closely linked to the genes conferring resistance to different pathogens and parasites. Amplified sequences can serve for the pro- duction of molecular markers for selection of resistant forms, or for molecular cloning of the novel resistance gene. The method discussed provided marking and mapping of the resistance genes in such cultivated plants as sunflower (downy mildew) [4], potato and tomato (Phytophtora and nematode) [10, 11], citrus plants, Citrus and Poncirus (virus and nematode of cit- rus) [12], melon (fusariose and aphid) [13], and soybean (virus, Phytophtora, and powdery mildew) [14, 15]. In the present study, a possibility of using the com- bination of primers designed from the conservative NBS domains of the genes RPS2 of Arabidopsis, N of tobacco, and L6 of flax, for amplification of the resis- tance-gene analogs from silverleaf sunflower (Helian- thus agrophyllus) was evaluated. The produced PCR products were cloned and characterized. Cloning and Analysis of the Resistance Gene Fragments from Silverleaf Sunflower Helianthus agrophyllus T. V. Danilova, M. Yu. Kuklev, G. N. Andreeva, V. S. Shevelukha, and G. I. Karlov Timiryazev Moscow Agricultural Academy, Moscow, 127550 Russia; fax: (495)977-84-28; e-mail: karlov@timacad.ru Received October 10, 2005; in final form, May 5, 2006 Abstract—Using a combination of degenerate primers designed from the NBS domains of the resistance genes, amplification and subsequent cloning of the resistance gene fragments from sunflower (Helianthus agro- phyllus) was conducted. Sequences of cloned PCR products differed from one another and displayed homology to NBS domain fragments of the already known plant resistance genes, as well as to the analogous genes from different classes. The highest homology was shown to the NBS domain regions of cultivated sunflower and the other members of the family Compositae. Two cloned fragments had open reading frames, while the other sequences carried stop codons and seemed to belong to pseudogenes. Amino acid sequences of Helianthus agrophyllus analyzed contained conservative regions typical of NBS domains of the resistance gene products. DOI: 10.1134/S1022795407040059 PLANT GENETICS