Identification of molecular markers linked to iron toxicity tolerance through bulk segregant analysis (BSA) in rice (Oryza sativa L.)

Abstract

Globally rice is the most important food crop, serving as staple food for more than half of the world’s population. As in other parts of the country, rice is the major food crop grown in Kerala too. The total annual production of rice is however insufficient to meet the total demand in the state. Iron toxicity prevalent in the rice growing tracts of the state, further compounds the problem of low rice production. Although, several attempts to ameliorate the iron toxic soil conditions are being made, the best way to combat this stress and increase rice production in the affected soils is to develop varieties tolerant to iron toxicity. The present investigation on 'Identification of molecular markers linked to iron toxicity tolerance through bulk segregant analysis (BSA) in rice (Oryza sativa L.)’ was conducted at College of Horticulture, Kerala Agricultural University (KAU), Vellanikkara, Thrissur during 2013 to 2015 year. The study involved screening of thirty rice genotypes for response to iron at toxic levels, hybridization between the most tolerant and susceptible genotype, production of F2 generation of this cross, parental polymorphism study using molecular markers and Bulk Segregant Analysis (BSA). The thirty rice genotypes were selected on the basis of their response to iron stress under KSCSTE project: ‘Donor identification for tolerance to iron toxicity in rice (Oryza sativa L.)’. Further screening of the thirty genotypes (Confirmation test 1 and 2) as per the method advocated by Shimizu et al. (2005) to confirm their tolerance or susceptibility to iron toxicity revealed existence of high significant differences among the genotypes with respect to leaf bronzing and biomass produced under varying concentrations of iron (0 ppm, 600ppm and 800ppm of Fe). Considering that at higher concentrations of Fe, a lower leaf bronzing and reduction in biomass, is a valid criterion for identifying genotypes tolerant to Fe stress, twelve genotypes viz., Cul-8709, Cul-210-29, AM-10-7, Cul-90-03, PM-709, ASD-16, ASD-18, Abhaya, IR-1552, T(N)-1, IR-36 and Cul-3 213 were found to be highly susceptible to iron stress while genotypes Cul-8723, Tulasi, Cul-18716, Kargi and IVT-33 were identified as the most tolerant ones. Selfing of F1s obtained on hybridizing the genotype (Tulasi) and genotype (CUL-8709) which were found respectively to be most tolerant and most susceptible to iron stress was done, to produce F2 population for the conduct of bulk sergegant analysis (BSA). Phenotyping of F2 plants under iron at toxic levels indicated presence of wide variability for shoot length, root length, total number of roots, number of fresh roots, shoot weight, root weight and visual scoring for iron-toxicity symptoms. The measures of skewness and kurtosis for various traits revealed a large quantitative variability. All the above traits except iron content in root of F2 lines exhibited a positive platykurtic distribution pointing to presence of gene interaction in trait expression. Measures of skewness and kurtosis also indicated occurrence of transgressive segregation in the F2 population. Leaf bronzing the typical symptom of Fe toxicity, showed a strong negative correlation with shoot length, root length, total number of roots, number of fresh roots, shoot weight and root weight. The results indicated that leaf bronzing is associated with growth reduction due to Fe2+ toxicity in this F2 population. Parental polymorphism (Tulasi and CUL-8709) survey using 338 Rice Microsatellites (RM) markers revealed 37 RM markers polymorphic between the two. These 37 polymorphic rice microsatellites markers (SSR markers) were found to be distributed over all 12 linkage groups of rice varying between one in case on Chromosome 7 to five each on Chromosome 2, 9 and 10. Bulk segregant analysis indicated that out of the 37 microsatellite markers that were polymorphic between parents seven viz., RM 263, RM 107, RM 12292, RM 24616, RM 24664, RM 13619 showed clear co-segregation with the susceptible parent and susceptible bulk, and resistant parent and resistant bulk. Probability of all seven putative markers was highly significant (P < 0.001) indicating strong association of these markers to the genomic region governing Leaf Bronzing Index which is a valid indicator of tolerance to iron toxicity. Through single marker analysis, three probable quantitative trait loci (QTL’s) of Leaf Bronzing Index were identified, each on 214 chromosome 1, 2 and 9. The QTL on chromosome 1 was located between 42.8 Mb and 43.2 Mb and associated with markers RM 12255 and RM 12292. The QTL for LBI was found to be associated with RM 13619 and RM 263 markers and placed between 24.9 Mb and 25.9 Mb on chromosome 2 while on chromosome 9, it was a located between 19.3 Mb and 20.1 Mb and linked to marker RM 107, RM 24616 and RM 24664.