Selection of stable reference genes for qPCR expression of Colletotrichum lindemuthianum, the bean anthracnose pathogen.

Phaseolus vulgaris L., commonly known as the common bean, is a highly nutritious crop often called the "poor man's meat". However, it is susceptible to various diseases throughout the cropping season, with anthracnose caused by Colletotrichum lindemuthianum being a significant threat that leads to substantial losses. There is still a lack of understanding about the molecular basis of C. lindemuthianum pathogenicity. The first step in understanding this is to identify pathogenicity genes that express more during infection of common beans. A reverse transcription quantitative real-time PCR (qPCR) method can be used for virulence gene expression. However, this approach requires selecting appropriate reference genes to normalize relative gene expression data. Currently, there is no reference gene available for C. lindemuthianum. In this study, we selected eight candidate reference genes from the available genome of C. lindemuthianum to bridge the gap. These genes were ACT (Actin), ß-tub (ß-tubulin), EF (Elongation Factor), Cyt C (Cytochrome C), His H3 (Histone H3), CHS1 (Chitin synthetase), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and abfA (Alpha-l-Arabinofuranosidase A). The primers for these candidate reference genes were able to amplify cDNA only from the pathogen, demonstrating their specificity. The qPCR efficiency of the primers ranged from 80% to 103%. We analyzed the stability of gene expression in C. lindemuthianum by exposing the mycelium to nine different stress conditions. We employed algorithms, such as GeNorm, NormFinder, BestKeeper, and RefFinder tools, to identify the most stable gene. The analysis using these tools revealed that EF, GAPDH, and ß-tub most stable genes, while ACT and CHS1 showed relatively low expression stability. A large number of potential effector genes have been identified through bioinformatics analysis in C. lindemuthianum. The stable genes for qPCR (EF and GAPDH) discovered in this study will aid the scientific community in determining the relative expression of C. lindemuthianum effector genes.

Fine mapping of a new common bean anthracnose resistance gene (Co-18) to the proximal end of Pv10 in Indian landrace KRC-5.

KEY MESSAGE: Mapping and fine mapping of bean anthracnose resistance genes is a continuous process. We report fine mapping of anthracnose resistance gene Co-18 which is the first anthracnose gene mapped to Pv10. The discovery of resistance gene is a major gain in the bean anthracnose pathosystem research. Among the Indian common bean landraces, KRC-5 exhibit high levels of resistance to the bean anthracnose pathogen Colletotrichum lindemuthianum. To precisely map the anthracnose resistance gene, we used a Recombinant Inbred Line (F2:9 RIL) population (KRC-5 × Jawala). The inheritance test revealed that KRC-5 carries a dominant resistance gene temporarily designated as Co-18. We discovered two RAPD markers linked to Co-18 among 287 RAPD markers. These RAPD markers were eventually developed into SCARs (Sc-OPR15 and Sc-OPF6) and flank Co-18 on chromosome Pv10 at a distance of 5.3 and 4.2 cM, respectively. At 4.0-4.1 Mb on Pv10, we detected a SNP (single-nucleotide polymorphism) signal. We synthesized 58 SSRs and 83 InDels from a pool of 135 SSRs and 1134 InDels, respectively. Five SSRs, four InDels, and two SCARs were used to generate the high-density linkage map, which led to the identification of two SSRs (SSR24 and SSR36) that are tightly linked to Co-18. These two SSRs flank the Co-18 to 178 kb genomic region with 13 candidate genes including five NLR (nucleotide-binding and leucine-rich repeat) genes. The closely linked markers SSR24 and SSR36 will be used in cloning and pyramiding of the Co-18 gene with other R genes to develop durable resistant bean varieties.