Bulked Segregant RNA Sequencing (BSR-Seq) Combined with SNP Genotyping Towards Mapping and Characterization of a Purple Blotch Resistance Gene in Onion (Allium cepa L.)

Abstract

The pathogenic fungus, Alternaria porri (Ellis) Cifferi, that causes purple blotch (PB) disease, is a major constraint to production of onion and allied crops worldwide. In the present study, bulk segregant RNA sequencing (BSR-Seq) was used to analyze onion cultivar Arka Kalyan (resistant parent), Agrifound Rose (susceptible parent), and two sets of their bulks (20 homozygous resistant and 20 susceptible) from F₆ RIL population to identify a potential region for resistance to PB. Transcript profiling resulted in 278.08 million clean reads from 8 libraries. Comparative expression analysis revealed 755 differentially expressed genes (DEGs) including 492 upregulated and 263 downregulated sequences. Bulk frequency ratio (BFR) was estimated between resistant and susceptible bulk, and 2963 common SNPs with BFR > 6 were detected on 1439 transcripts. Euclidean distance association analysis identified a 7.3 Mb resistance specific candidate region in the long arm of chromosome 6. Using RNA-Seq, 23 DEGs were reported in the candidate region in chromosome 6, including ACCL_20794 (Chr6: 187,639,724–187,643,297), a disease-resistant protein of the CC-NBS-LRR class, whose expression was elevated in the resistant pools following PB treatment. The ACCL_20794 gene was cloned and based on the sequences from the two parents, a single amino acid mutation—histidine (H) to serine (S) was detected in the resistance genotype Arka Kalyan. Quantitative reverse transcription (qRT)-PCR further demonstrated significantly differential expression of ACCL_20794 in the two parents as well as the RIL bulks. This indicates that ACCL_20794 might be the candidate resistance gene ApR1 and is implicated in the PB resistance response.