Molecular Mapping Of QTLs Conferring Resistance To Fusarium Root Rot In Castor Bean (Ricinus Communis)

ObjectiveThe plant disease resistance trait is of much importance to plant breeders.Thus,the advent of Quantitative Trait Loci?QTL?mapping has proven to be much significant,by reducing the stress breeders used to go through in classical breeding.Therefore,researchers have identified the genes conferring quantitative traits to many plants through QTL mapping.Fusarium root rot caused by Fusarium solani species complex is a serious disease of castor?Ricinus communis?,especially in China.This disease is reported to have affected over?1800?0.66 ha of castor plants in 2016 in an outbreak in South China.Presently,not much research has been done in identifying QTLs linked to the resistance to fusarium root rot or Fusarium spp diseases in castor.Hence,identifying DNA markers linked to the resistance of castor to this disease will be much beneficial to castor breeders in efficiently developing resistant cultivars.After the identification of QTLs,the following step is to identify the genes conferring the resistance to fusarium root rot.These traits can be engineered into castor plants to aid in its resistance to fusarium root rot.Materials and MethodsTo identify QTLs for resistance to fusarium root rot,a mapping population of 260 F₂progenies,derived from disease-resistant and disease-susceptible parents was designed.The mapping population was planted at the greenhouse in autumn 2017.The fusarium root rot pathogen was cultured at the plant pathology laboratory of Guangdong Ocean University and was artificially inoculated into the young castor seedlings.Disease incidence was scored based on the days taken for the plants to wilt.Highly resistant plants that showed no wilt after the end of the experiment were given a score of 1 and highly susceptible plants which wilted within the first 30 days were given a score of 8.Generally,the phenotypic score was from 1-8.DNA from the young leaves were also extracted.Already existing SSR primers designed by our laboratory were screened for polymorphism for resistance using the Bulked Segregant Analysis?BSA?and polymorphism was confirmed by randomly selecting few individuals from each extreme and amplifying them with the selected markers.The confirmed polymorphic markers for resistance to fusarium root rot were used to scan the entire F₂ population and were genotyped.The SSR marker genotypes were used to construct a linkage map.Data from the phenotypic analysis and the linkage map were combined to draw a QTL map to identify the particular area on the linkage map with the trait of interest.After the QTL was identified,the scaffolds of the SSR markers within the QTL region were juxtaposed with the already published genome of castor on the National Centre for Biotechnology Information to identify the linkage of these markers to any genes.ResultsThe in vivo pathogenicity test revealed a highly skewed but continuous frequency distribution.The frequency of resistance did not follow a normal distribution,indicating the control of a major QTL in resistance to fusarium root rot.Out of 400 SSR primers screened under the BSA method,96 were polymorphic for resistance.Further confirmatory screening of the 96 primers identified 11 highly polymorphic primers for resistance.The 11 SSR markers were scanned with the 260 F₂ individuals.The 11 SSR markers were then genotyped and scored according to the software QTL IciMapping.The scored values were entered into the QTL IciMapping software using the MAP module at a LOD threshold of 5 to construct the linkage map.One linkage map was drawn covering 51.04cM of the genome and having an average interval of 4.64cM.A combination of the linkage map drawn and the phenotype data was used to detect QTLs on the map using the composite interval method of QTL mapping.From the analysis,the software was able to identify one QTL?FRR?on the chromosome?Linkage group?.qFRR was located at a position of 34cM between primers RCM1471 and RCM1311 having a phenotypic variation of greater than 10 and having a LOD value of 7.58.Analysis of the scaffold regions of the primers?RCM 1471 and RCM 1311?within the QTL region with conserved amino sequences of the castor genome on?National Centre for Biotechnology Information?NCBI BLAST discovered two putative genes,“LOC8268784cysteine proteinase inhibitor”and“LOC8281835 cell division cycle and apoptosis regulator protein”.The cysteine proteinase inhibitor belongs to the cystatin superfamily which has been identified in humans and other plants to confer antimicrobial properties.The cell division cycle and apoptosis regulator 1 gene is also known as the CCAR1 or p30 dbc gene has been known in humans to control tumorigenesis and has been studied in the treatment of cancer.ConclusionFrom the phenotypic distribution,we inferred that the resistance to fusarium root rot in castor in our experiment was controlled by a major QTL,which was confirmed from the one major QTL?LOD=7.58;PVE%=13.80?that was linked through the QTL mapping method.The identification of this QTL is essential in castor breeding for resistance to fusarium root rot since it is the first of its kind.Furthermore,the results from the analysis of the scaffold of the markers RCM1311 and1471 revealed the existence of two putative genes which after further tests will be critical in castor disease resistance breeding.