Genetic Diversity Of Common Wild Rice And NBS-LRR Resistance Gene Exploring

Common wild rice(Oryza rufipogon Griff.) is an important gene resource for Asian cultivated rice(O. sativa L.), and have abundant genetic diversity and various resistant genes. Due to the habitat shrinkage, the number and acreage of wild rice are decreasing. It is of paramount important to evaluate the genetic diversity and developed core collections of wild plants to conserve the genetic resources harboring resistant genes, and for their efficient use in breeding strategies. In the present study, the morphological and SSR(Simple sequence repeats) markers were used to investigate the common wild rice from eight populations(Dongxiang, Fogang, Boluo, Zengcheng, Huilai, Gaozhou, Suixi and Qionghai). The phenotypic and genotypic data were used to evaluate the genetic diversity and develop the core collection. Then, whole genome re-sequencing of two accessions was done, for exploring the NBS-LRR like mutant genes. The results were as follow:We evaluated the genetic diversity of these accessions by 13 phenological traits, and genotyped them for 36 DNA markers evenly distributed on the 12 chromosomes. The CV(Coefficient of variation) of quantitative traits was 0.56 and ranged from 0.37 to 1.06, and the secondary branch showed the highest CV(2.46). Boluo population showed the highest phenotypic variation, and Fogang exhibited the lowest. The qualitative traits showed some correlation with the latitude of wild species. The number of effect alleles(ne), Nei’s gene diversity(h), Shannon’s Information index(I) and polymorphism information content(PIC) were 1.403, 0.239, 0.365 and 0.640, respectively indicating that the marker loci have a high level of polymorphism and genetic diversity in all populations.,. Zengcheng, Gaozhou and Suixi populations possessed the higher levels of genetic diversity, whereas the Huilai and Boluo populations have lower levels of genetic diversity than other populations. Genetic differentiation and gene flow revealed low gene exchanged between eight populations, and Zengcheng population revealed the highest difference compared to other population. Phylogenetic analyses based on morphological and molecular data revealed remarkable differences in the genetic diversity of common wild rice populations. Based on their genetic distance of phenotypic and genotypic data, 130 accessions were selected as a core collection from entire populations, and the core collections hold the 12.1-20% germsplm in entire populations. According to the phenotypic validation, quantitative traits showed non-significant difference between entire populations and core collections, and the qualitative traits represented the 100% of entire populations. The genotypic validation showed that there was no repeated genotype in all core collections, and the core collections could cover the all groups in phenotypic and genotypic dendrograms, and uniformly distributed in PCA(Principal component analysis). The core collections retained 94.2-98.82% of the alleles in entire populations. The genetic diversity parameters revealed non-significant differences between entire populations and core collections. This genetically diverse core collection had good representativeness, and retained higher genetic diversity.Two self-crossed lines from core collection were re-sequenced by Illumina Hi Seq 2000 100 PE high-throughput platform, and then compared with the reference genome of 93-11 and Nipponbare cultivars. The results showed that coverage depth(>=1x) was over 89.59% and mean coverage depth was above 32.48 x. DXW102 was close to Nipponbare, and DXW103 was more similar to 93-11 based on SNP and In Del dataset. The genes annotations for SNP and In Del showed that DXW103 had the most variation for Nipponbare reference genome, and DXW102 had the least variation for Nipponbare. A total of 53.1% and 55.1% common genes were detected in two lines compared to the reference genome of 93-11 and Nipponbare cultivars, respectively.We screened out NBS-LRR like mutant candidate genes based on gene annotation and 100% of the explored NBS genes could be validated by PCR. The NBS analysis of structure, distribution on chromosomes and sequences diversity showed that NBS domain were more conserved than LRR domain; NBS-LRR was mainly distributed on chromosome 11. The genetic diversity of NBS was ranged from 2.565 to 6.701, and the NBS diversity of DXW102 was higher than DXW103. Furthermore, the gene-gene interactions of NBS were analyzed, and the results showed that NBS gene interacted mostly with the cytochrome C. All the data showed that common wild rice had abundant NBS-LRR mutant genes, and the method of comparing with two reference genomes would be more useful in NBS gene exploration.This study evaluated the genetic diversity and developed a core collection of common wild rice. Then, the NBS-LRR mutant genes were explored by re-sequencing of two lines, and the structure and function of NBS were analyzed. These works would supply the new breeding resources and favorable mutant genes for the breeding of resistant rice.