Genome wide re-sequencing of newly developed Rice Lines from common wild rice (Oryza rufipogon Griff.) for the identification of NBS-LRR genes

Abstract

Common wild rice (Oryza rufipogon Griff.) is an important germplasm for rice breeding, which contains many resistance genes. Re-sequencing provides an unprecedented opportunity to explore the abundant useful genes at whole genome level. Here, we identified the nucleotide-binding site leucine-rich repeat (NBS-LRR) encoding genes by re-sequencing of two wild rice lines (i.e. Huaye 1 and Huaye 2) that were developed from common wild rice. We obtained 128 to 147 million reads with approximately 32.5-fold coverage depth, and uniquely covered more than 89.6% (> = 1 fold) of reference genomes. Two wild rice lines showed high SNP (single-nucleotide polymorphisms) variation rate in 12 chromosomes against the reference genomes of Nipponbare (japonica cultivar) and 93-11 (indica cultivar). InDels (insertion/deletion polymorphisms) count-length distribution exhibited normal distribution in the two lines, and most of the InDels were ranged from -5 to 5 bp. With reference to the Nipponbare genome sequence, we detected a total of 1,209,308 SNPs, 161,117 InDels and 4,192 SVs (structural variations) in Huaye 1, and 1,387,959 SNPs, 180,226 InDels and 5,305 SVs in Huaye 2. A total of 44.9% and 46.9% genes exhibited sequence variations in two wild rice lines compared to the Nipponbare and 93-11 reference genomes, respectively. Analysis of NBS-LRR mutant candidate genes showed that they were mainly distributed on chromosome 11, and NBS domain was more conserved than LRR domain in both wild rice lines. NBS genes depicted higher levels of genetic diversity in Huaye 1 than that found in Huaye 2. Furthermore, protein-protein interaction analysis showed that NBS genes mostly interacted with the cytochrome C protein (Os05g0420600, Os01g0885000 and BGIOSGA038922), while some NBS genes interacted with heat shock protein, DNA-binding activity, Phosphoinositide 3-kinase and a coiled coil region. We explored abundant NBS-LRR encoding genes in two common wild rice lines through genome wide re-sequencing, which proved to be a useful tool to exploit elite NBS-LRR genes in wild rice. The data here provide a foundation for future work aimed at dissecting the genetic basis of disease resistance in rice, and the two wild rice lines will be useful germplasm for the molecular improvement of cultivated rice.

Fig 1. Common wild rice lines at reproductive stage in the field.

(A) Huaye 1. (B) Huaye 2. Right panels are representing grains of respective lines. These lines were developed from non-shattering plant with high seed setting of common wild rice.

Fig 2. Annotation and distribution of SNPs and InDels between common wild rice lines and Nipponbare reference genome.

SNPs, insertions and deletions on the rice pseudomolecules were classified as genic and intergenic regions, and locations within the gene models were annotated. The number of SNPs, insertions and deletions in each class is shown. A and B indicate the distributions of SNPs and InDels in the comparisons of Huaye 1 and Nipponbare reference genome, and Huaye 2 and Nipponbare reference genome, respectively.

Fig 3. Functional characterization of the resistance genes into gene ontology (GO) terms.

(A) and (B) indicate the annotation of resistance genes that were detected in the comparisons of Huaye 1 and reference genomes, and Huaye 2 and reference genomes of Nipponbare and 93–11, respectively.

Fig 4. Validation of NBS like genes in wild rice genome.

(A) and (B) indicate the PCR products of Huaye 1 and Huaye 2 mapped onto the reference genomes, respectively. M represents DNA Marker, and the vertical letters indicate the gene ID.

Fig 5. Density map of NBS-LRR resistance genes that were detected between Huaye 1 and reference genome of 93–11.

Fig 6. Phylogenetic relationships between NBS regions in two wild rice lines.

The names in each branch represent wild rice lines, chromosome number and gene ID. *indicates the novel NBS like genes that were explored from particular reference genome. The scale bar indicates the genetic distance.

Fig 7. Protein-protein interaction network of NBS-related genes associated with the resistant factors.

(A) and (B) indicate the protein-protein interaction network of NBS-related genes between wild rice lines and reference genome of 93–11, (C) indicates the protein-protein interaction network of NBS-related genes between wild rice lines and reference genome of Nipponbare.