Mining Genome and Transcriptome Sequences of Poncirus trifoliata for NB-LRR Genes and Their Association with HLB Disease Resistance

Nucleotide binding site-leucine rich repeat (NB-LRR)  genes are among the most important gene classes for plant breeding and genetic engineering purposes being responsible for activation of plant defense mechanisms against various pathogens. The bacterial disease Huanglongbing (HLB) is a most devastating disease of citrus worldwide. Several studies have indicated that Poncirus is resistant/tolerant to HLB, but the Poncirus genes for HLB resistance/tolerance are to be identified and characterized. As of today, no report exists on sequence and expression analysis of NB-LRR gene repertoire in Poncirus. In the present study, RNA-seq and DNA-seq paired end reads were generated using Illumina HiSeq 2000 and aligned to 618 nucleotide sequences of recently characterized NB-LRR genes from the Citrus clementina genome using TopHat and BWA, respectively. We found ~1.45 million reads from DNA-seq data matching to 616 NB-LRR related genes from the C. clementina genome. These Poncirus NB-LRR sequences showed an overall pairwise identity of 84.8-99.5% and 68.9-99.3% identical site with the NB-LRR genes of C. clementina genome. We identified structural variations and SNP/indels in the coding sequences of these genes. RNAseq data analysis identified 208 contigs matching with 141 NB-LRR genes in non-inoculated Poncirus, while 158 contigs corresponding to 112 NB-LRR genes in HLB-inoculated Poncirus. We identified that 75 genes were up-regulated (>2 fold) and 63 genes were down-regulated (<2 fold) in HLB-inoculated Poncirus. Differentially expressed NB-LRR isoforms in HLB-inoculated Poncirus were also detected. The identified Poncirus NB-LRR gene repertoire would assist in the study of their functions in HLB disease resistance.