Búsqueda, selección y localización genética de marcadores análogos a genes de resistencia en poblaciones de avena segregantes para caracteres de resistencia a la roya de la corona

Abstract

Plants have developed a precise system to defense against pathogens attack. However, these are able to escape the different strategies of plant defense leading to disease development, with important impact on the productivity of agronomical interest crops. Therefore, one of the main objectives of plant breeding is the development of varieties resistant to pathogens as the most effective method of disease control. In Avena, the development of new resistant cultivars to the fungus Puccinia coronata, causal agent of crown rust, or resistant cultivars to other disease-causing fungi is the most effective method to control these diseases. However, the major disadvantage is the time required for their production. An appropriate instrument to shorten these processes is the use of markers to perform marker-assisted selection. Increasing knowledge of gene sequences involved in resistance to pathogens allows the collection of markers based on these sequences. These functional markers have advantages over neutral molecular markers, including the greater likelihood of identifying candidate genes responsible for the phenotypic manifestation of resistance. The molecular structure of proteins encoded by resistance genes to pathogens isolated from different species is characterized by the presence of conserved domains. These have facilitated the isolation of similar sequences to these genes in different plant species. These sequences are known as analogues resistance genes or RGAs. The RGAs have become efficient markers of resistance traits controlled by qualitative genes and QTLs. In this paper, we have used different methodologies for RGAs markers in oat. One of them has used RGAs sequences isolated from Avena species and other species near them, such as wheat and barley, as markers for RFLPs. The existence of a large number of expressed sequences in these related species allows the selection of sequences with RGAs features. This has facilitated the collection of homologous markers in oat. The second methodology used consisted of a multilocus procedure, called NBS/PK profiling, developed with degenerate primers targeting conserved motifs of R genes, like the conserved motifs of the NBS-LRR class and the protein kinases, also involved in resistance against pathogens. We proceeded to the genetic location of all types of markers used in three genetic maps of oat. A map derived from the cross between two diploid species A.strigosa x A.wiestii that segregates for two major resistance genes to P. coronata, a second map between two genotypes of the hexaploid species A. sativa (MN841801-1 x 'Noble-2') that segregates for eight QTLs for partial resistance to P. coronata, and a third map which is considered the reference map from the crossing hexaploid A. byzantina cv ‘Kanota’ and A. sativa cv ‘Ogle’. In this paper, have been identified markers linked to major resistance genes and QTLs segregating in the populations, and markers located in regions where others resistance loci have been identified in different oat maps. RGAs probes used for RFLP markers identified both homologous and homoeologus loci in polyploid species. These could be a disadvantage to provide appropriate markers for each specific resistance locus. We have developed a procedure to convert RGA-RFLP markers in locus-specific markers or sequence tagged site (STS) markers. Throughout the evolution of the genus Avena have been a large number of chromosomal rearrangements, which have hindered to establish of homology and homoeology relationships between chromosomes of different species and within polyploid species, respectively. And these have also affected the establishment of relationships between linkage groups. In this paper, the common RGA-RFLP markers located in different genetic maps have allowed to confirm and establish some of the homoeology and homology relationships between linkage groups. The chromosomal rearrangements within the genus have prevented the construction of genetic maps with an equivalent number of linkage groups and chromosomes and relate each linkage group with its corresponding chromosome. In this work we have used monosomic series of hexaploid oat and RGAs markers, and we have followed cytogenetic procedures to solve these problems. First we have characterized the monosomic series of A. byzantina cv ‘Kanota’ and A. sativa cv ‘SunII’, using repetitive sequence specific of genome and ribosomal DNA as FISH probes. These have allowed the identification of different chromosomes of the hexaploid complement and unify the nomenclatures used to call them by different investigators. Secondly, we have used chromosome microdissection of monosomic lines for extracting DNA of the univalent chromosome in cells in metaphase I. The subsequent DNA amplification of RGAs that previously identified in the maps of oats has established relationship between chromosomes and linkage groups. Furthermore, FISH experiments have been performed with RGAs probes previously located on oat maps, which has made it possible to establish relationships between linkage groups and chromosomes in different species of Avena.