Sequence haplotypes revealed by sequence-tagged site fine mapping of the Ror1 gene in the centromeric region of barley chromosome 1H

Abstract

We describe the development of polymerase chain reaction-based, sequence-tagged site (STS) markers for fine mapping of the barley (Hordeum vulgare) Ror1 gene required for broad-spectrum resistance to powdery mildew (Blumeria graminis f. sp. hordei). After locating Ror1 to the centromeric region of barley chromosome 1H using a combined amplified fragment length polymorphism/restriction fragment-length polymorphism (RFLP) approach, sequences of RFLP probes from this chromosome region of barley and corresponding genome regions from the related grass species oat (Avena spp.), wheat, and Triticum monococcum were used to develop STS markers. Primers based on the RFLP probe sequences were used to polymerase chain reaction-amplify and directly sequence homologous DNA stretches from each of four parents that were used for mapping. Over 28,000 bp from 22 markers were compared. In addition to one insertion/deletion of at least 2.0 kb, 79 small unique sequence polymorphisms were observed, including 65 single nucleotide substitutions, two dinucleotide substitutions, 11 insertion/deletions, and one 5-bp/10-bp exchange. The frequency of polymorphism between any two barley lines ranged from 0.9 to 3.0 kb, and was greatest for comparisons involving an Ethiopian landrace. Haplotype structure was observed in the marker sequences over distances of several hundred basepairs. Polymorphisms in 16 STSs were used to generate genetic markers, scored by restriction enzyme digestion or by direct sequencing. Over 2,300 segregants from three populations were used in Ror1 linkage analysis, mapping Ror1 to a 0.2- to 0.5-cM marker interval. We discuss the implications of sequence haplotypes and STS markers for the generation of high-density maps in cereals.

Figure 1

Haplotype structure of Ror1-linked marker sequences. Ror1-linked marker sequences were determined from each of the four parental barley lines BCIngrid mlo-5 (I), Malteria Heda (M), BCPallas mlo-5 (P), and Grannenlose Zweizeilige (G). Segments of the sequence defined by the polymorphic bases (tick marks) were classified as being the same as BCIngrid mlo-5 (clear) or of a second type (colored) according to the nucleotides present at the polymorphic sites. The eight sequences shown are those for which two or more polymorphic sites were found and for which all four barley lines were assayed for all polymorphic sites. The interruption in the CDO1188 figure represents 1,200 bp of sequence containing no polymorphic sites.

Figure 2

Genetic maps of the centromeric region of barley chromosome 1H constructed using three Ror1 mapping populations. Distances are shown in centiMorgans. Dotted lines join markers common between the maps.