Genomic organization, rapid evolution and meiotic instability of nucleotide-binding-site-encoding genes in a new fruit crop, "chestnut rose"

Abstract

From chestnut rose, a promising fruit crop of the Rosa genus, powdery mildew disease-resistant and susceptible genotypes and their F(1) progeny were used to isolate nucleotide-binding-site (NBS)-encoding genes using 19 degenerate primer pairs and an additional cloning method called overlapping extension amplification. A total of 126 genes were harvested; of these, 38 were from a resistant parent, 37 from a susceptible parent, and 51 from F(1) progeny. A phylogenetic tree was constructed, which revealed that NBS sequences from parents and F(1) progeny tend to form a mixture and are well distributed among the branches of the tree. Mapping of these NBS genes suggested that their organization in the genome is a "tandem duplicated cluster" and, to a lesser extent, a "heterogeneous cluster." Intraspecific polymorphisms and interspecific divergence were detected by Southern blotting with NBS-encoding genes as probes. Sequencing on the nucleotide level revealed even more intraspecific variation: for the R4 gene, 9.81% of the nucleotides are polymorphic. Amino acid sites under positive selection were detected in the NBS region. Some NBS-encoding genes were meiotically unstable, which may due to recombination and deletion events. Moreover, a transposon-like element was isolated in the flanking region of NBS genes, implying a possible role for transposon in the evolutionary history of resistance genes.

Figure 1.—

Phylogenetic position of chestnut rose in higher plant taxa. The flower and fruit of the chestnut rose are shown. Modified from Figure 1 of Ku et al. (2000).

Figure 2.—

A transposon-like element was isolated close to the NBS domain. (A) The strategy for isolating flanking sequences, for which an adaptor was designed. Arrows show the location of the PCR primers. (B) PCR products of three rounds (I, II, and III) of TAIL–PCR. (C) The sequence was highly homologous to a transposon. (D) RFLP pattern confirmed the gene as a transposon and polymorphism was detected among crops in Rosaceae. I, negative control; II, resistant parent; III, susceptible parent; IV, R. roxburghii cv. Guinong no.1; V, R. sterilis; VI, Prunus persica; VII, Pyrus communis; VIII, Malus baccata.

Figure 3.—

Phylogenetic analysis and genetic mapping of chestnut rose NBS genes. The tree was constructed by the neighbor-joining method with human APAF1 as outgroup by PAUP* 4.0 software. Different colors denote different sources. Bootstrap values (1000 replicates) with only values >50% are shown on the branches. (Right) Three clusters were obtained by MAPMAKER and Map Manager QTXb20 software. The phylogenetic position of some RGH genes is connected to their locations in the genetic map by lines, illustrating two forms of cluster in the chestnut rose genome: the “tandem duplicated cluster,” genes that occupy the same phylogenetic lineage within a cluster, and, conversely, the “heterogeneous cluster.”

Figure 4.—

Rapid evolution of NBS-encoding genes in chestnut rose. (A) Intraspecific variation of copy number and size between resistant (R) and susceptible (S) parents. (B) Interspecific polymorphisms in Rosaceae family. (Top) A rapidly evolving gene. (Bottom) A rather old gene, which shows a uniform band among different species in Rosaceae. (C) Nucleotide polymorphism detected among different species. G6, G5, and G1 denote different cultivars in R. roxburghii; WZ, R. sterilis; and rose, R. chinensis.

Figure 5.—

Meiotic instability of NBS-encoding genes. (A) A new homolog was produced in F₁ progeny. (B) Homologs of different sizes were produced in F₁ individuals. (C) Deletion was detected in genes from F₁ progeny.