Mapping QTLs for Salt Tolerance in Rice (Oryza sativa L.) by Bulked Segregant Analysis of Recombinant Inbred Lines Using 50K SNP Chip

Abstract

Soil salinity is a major constraint to rice production in large inland and coastal areas around the world. Modern high yielding rice varieties are particularly sensitive to high salt stress. There are salt tolerant landraces and traditional varieties of rice but with limited information on genomic regions (QTLs) and genes responsible for their tolerance. Here we describe a method for rapid identification of QTLs for reproductive stage salt tolerance in rice using bulked segregant analysis (BSA) of bi-parental recombinant inbred lines (RIL). The number of RILs required for the creation of two bulks with extreme phenotypes was optimized to be thirty each. The parents and bulks were genotyped using a 50K SNP chip to identify genomic regions showing homogeneity for contrasting alleles of polymorphic SNPs in the two bulks. The method was applied to 'CSR11/MI48' RILs segregating for reproductive stage salt tolerance. Genotyping of the parents and RIL bulks, made on the basis of salt sensitivity index for grain yield, revealed 6,068 polymorphic SNPs and 21 QTL regions showing homogeneity of contrasting alleles in the two bulks. The method was validated further with 'CSR27/MI48' RILs used earlier for mapping salt tolerance QTLs using low-density SSR markers. BSA with 50K SNP chip revealed 5,021 polymorphic loci and 34 QTL regions. This not only confirmed the location of previously mapped QTLs but also identified several new QTLs, and provided a rapid way to scan the whole genome for mapping QTLs for complex agronomic traits in rice.

Fig 1. Frequency distribution of SSI for grain yield among CSR11/MI48 RILs under.

(A) Moderate sodicity and (B) High sodicity.

Fig 2. Analysis of heterogeneous loci in different RIL pool sizes and mixture of parental DNA samples using 50K SNP chip.

Genomic DNA from 10 to 50 individual RILs of CSR11/MI48 mapping population were pooled in equal amounts, with higher pools including all the RILs of lower pools for the analysis of allele heterogeneity (Red line). Computational expectations on Bootstrap analysis of the pools of 10 to 50 lines, showing successive increase in heterogeneity up to 94% (Green line). Observed heterogeneity with mixing of genomic DNA from the two parental lines in the proportions of 1:5, 1:4, 1:3, 1:2 and 1:1 (Blue line).

Fig 3. Physical map positions of QTLs identified by BSA of CSR11/MI48 RIL population using 50K SNP chip.

QTLs shown in green color have salt tolerant allele coming from the tolerant parent CSR11 and those in red color have tolerant allele contributed by the sensitive parent MI48.

Fig 4. QTL positions identified in CSR27/MI48 population by BSA using 50k SNP chip.

Physical map position of QTLs with green color showing tolerant allele coming from tolerant parent CSR27 (11 loci), red color showing tolerant allele coming from sensitive parent MI48 (23 loci). Blue and violet bars represent earlier identified QTLs by Ammar et al. [42] and Pandit et al. [8], respectively.