Mapping of STS markers obtained from oat resistance gene analog sequences

Abstract

Two previously isolated resistance gene analogs (RGAs) of oat have been located as RFLPs in the reference map of Avena byzantina 'Kanota' x Avena sativa 'Ogle' in regions either homologous or homoeologous to loci for resistance to Puccinia coronata, the causal agent of crown rust. In this study, the RGAs were mapped in two recombinant inbred line (RIL) populations that segregate for crown rust resistance: the diploid Avena strigosa x Avena wiestii RIL population (Asw), which has been used for mapping the complex locus PcA, and the hexaploid MN841801-1 x Noble-2 RIL population (MN), in which QTLs have been located. To obtain single-locus markers, RGAs were converted to sequence tagged site (STS) markers using a procedure involving extension of the original RGA sequence lengths by PCR genome walking, amplification and cloning of the parental fragments, and identification of single nucleotide polymorphisms. The procedure successfully obtained STSs from different members of the L7M2 family of sequences, the initial NBS of which have nucleotide similarities of >83%. However, for RGA III2.18, the parental lines were not polymorphic for the STSs assayed. A sequence characterized amplified region (SCAR) marker with features of an RGA had been previously identified for gene Pc94. This marker was also mapped in the above RIL populations. Markers based on RGA L7M2 co-localized with markers defining the QTL Prq1a in linkage group MN3, and were located 15.2 cM from PcA in linkage group AswAC. The SCAR marker for Pc94 was also located in the QTL Prq1a but at 39.5 cM from PcA in AswAC, indicating that the NBS-LRR sequence represented by this marker is not related to PcA. L7M2 was also excluded as a member of the PcA cluster, although it could be an appropriate marker for the Prq1a cluster if chromosome rearrangements are postulated.

Use of Tyramide-Fluorescence in situ Hybridization and Chromosome Microdissection for Ascertaining Homology Relationships and Chromosome Linkage Group Associations in Oats

“…The L7M2 RGA family of A. strigosa is described as being composed of at least 4 members of the nonTIR-NBS-LRR class of R genes [Irigoyen et al, 2004;Loarce et al, 2009]. Cloning and sequencing of fragments derived from 2 members of this family, L7M2.3 and L7M2.4, as described below, did not increase this postulated number of L7M2 family members.…”

“…III2.18 A. strigosa RGA III2.18 is a member of a nonTIR-NBS-LRR family of putative R genes. In A. strigosa , 3 highly similar sequences of 2100 bp have been previously isolated [Loarce et al, 2009]. However, the number of members of the family is likely more than 3, since at least 3 other related sequences, as described below, have been detected in A .…”

“…For tyramide-FISH (Tyr-FISH), 3 A. strigosa RGA probes, namely III2.18, L7M2.2-L7M2.3 and II2.17 belonging to 3 families of low copy number genes, were used [Irigoyen et al, 2004;Loarce et al, 2009]. III2.18 is 2,100 bp in length, L7M2.2 and L7M2.3 are sequences from 2 members of the same RGA family with lengths of 2,000 and 4,400 bp, respectively, and II2.17 is a sequence of 3,030 bp (GenBank accessions AJ296001, AJ619774, AJ619775, and JN121383, respectively).…”

“…Single base extension fragments were monitored by capillary electrophoresis in an ABI 3130XL Genetic Analyzer (Applied Biosystems). Successful SNPs for L7M2 were scored in a sample of 20 RILs from each mapping population and the obtained segregations compared with the previously mapped RFLP and STS markers for the L7M2 sequence [Irigoyen et al, 2004;Loarce et al, 2009].…”

“…FISH probes for the resistance genes rp1 and rp3 and the ␣ -zeinA gene family have been located at single loci in maize chromosomes [Kato et al, 2004]. This approach could be tried in oats since several sequences structurally related to resistance genes -resistance gene analogues (RGA) -have been cloned and their locations noted on diploid and hexaploid oat maps [Irigoyen et al, 2004;Loarce et al, 2009].…”