Large intraspecific haplotype variability at the Rph7 locus results from rapid and recent divergence in the barley genome

Abstract

To study genome evolution and diversity in barley (Hordeum vulgare), we have sequenced and compared more than 300 kb of sequence spanning the Rph7 leaf rust disease resistance gene in two barley cultivars. Colinearity was restricted to five genic and two intergenic regions representing <35% of the two sequences. In each interval separating the seven conserved regions, the number and type of repetitive elements were completely different between the two homologous sequences, and a single gene was absent in one cultivar. In both cultivars, the nonconserved regions consisted of approximately 53% repetitive sequences mainly represented by long-terminal repeat retrotransposons that have inserted <1 million years ago. PCR-based analysis of intergenic regions at the Rph7 locus and at three other independent loci in 41 H. vulgare lines indicated large haplotype variability in the cultivated barley gene pool. Together, our data indicate rapid and recent divergence at homologous loci in the genome of H. vulgare, possibly providing the molecular mechanism for the generation of high diversity in the barley gene pool. Finally, comparative analysis of the gene composition in barley, wheat (Triticum aestivum), rice (Oryza sativa), and sorghum (Sorghum bicolor) suggested massive gene movements at the Rph7 locus in the Triticeae lineage.

Figure 1.

Establishment of a 350-kb Physical Contig at the Rph7 Locus in the Resistant Cultivar Cebada Capa. (A) Genetic map of the Rph7 locus on chromosome 3HS (Brunner et al., 2003). (B) Comparison of the two physical maps in the susceptible cultivar Morex (Brunner et al., 2003) and the resistant cultivar Cebada Capa. Boxes represent the genes located at the Rph7 locus. White boxes correspond to the three genes (Hvgad1, Hvpg1, and Hvpg4) that were used for screening the Cebada Capa BAC library. The asterisk represents a BAC-end probe that was derived from BAC 58F1 and was used to establish the connection between BACs 124A1 and 14E11. The region spanning the Rph7 gene between the distal and proximal recombination break points (indicated with black vertical arrowheads) is shown as a dotted line.

Figure 2.

Sequence Rearrangements in the Intergenic Regions in Morex and Cebada Capa. (A) Schematic representation of 53 kb of DNA sequence corresponding to the assembly of the seven conserved regions (CR1-7) in Morex and Cebada Capa. The top line (M) represents the 53 kb of sequence from Morex and the bottom one (CC) the sequence from Cebada Capa. Filled boxes with color codes given in the figure represent transposable elements that have been inserted in each sequence. Hatched boxes represent nonrepetitive sequences that also differ from the common sequence stretch. Red triangles represent miniature inverted-repeat transposable elements. Genes are represented as black boxes and identified by name. The size scale for the conserved regions (CR1-CR7) is indicated as a line and is three times larger than the one (indicated as a box) used for the nonconserved repetitive and nonrepetitive sequences. Two small arrowheads indicate the duplicated region of 804 bp found near the 3′ end of HvHGA2 in Cebada Capa. (B) Model for the evolution of the Hvpg1-Hvpg4 intergenic region (CR3-CR4) in Cebada Capa (left) and Morex (right). Color codes are the same as in (A). Filled arrowheads indicate the insertion of an element, whereas empty ones indicate unequal recombination between LTR of the same elements leading to solo LTRs with identical TSDs.

Figure 3.

Haplotype Combinations Found in the Hvpg1-Hvpg4-HvHGA2 Intergenic Regions in 41 Cultivated Barley Lines. (A) Schematic representation of the Hvpg1-Hvpg4 and Hvpg4-HvHGA2 intervals in Morex (M) and Cebada Capa (CC) showing the position, orientation, and names of the primers used in the analysis. (B) PCR products amplified by four primer combinations and schematic representation of the six haplotype combinations identified in the 41 barley lines. The ITS14-15 primer combination was used as a positive control for PCR amplification. PCR products are represented as lines between the primers with their size above it.

Figure 4.

Orthologous Relationships between the Genes Located at the Rph7 Locus in Barley, Wheat, Rice, and Sorghum. The location of the genes on genetic maps and/or on physical maps is indicated on the left side of the chromosomes. Hv, Hordeum vulgare; Ta, Triticum aestivum; Sb, Sorghum bicolor; Os, Oryza sativa. The inversion identified by Klein et al. (2003) between rice chromosome 1 and sorghum chromosome 3 is indicated with dashed lines. A, B, and C indicate groups of synteny between at least two of the grass species investigated.