Computational protocol: Detection and verification of QTLs associated with heat-induced quality decline of rice (Oryza sativa L.) using recombinant inbred lines and near-isogenic lines

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Protocol publication

[…] Total DNA was extracted from the leaves by the CTAB method (). PCR was performed in a 5-μL reaction mixture containing 20 ng of genomic DNA, 2.0 μM forward and reverse primers and 2.5 μL of GoTaq Green Master Mix (Promega, USA). The amplification profile was 5 min at 94°C; 35 cycles of 45 s at 94°C and 1.5 min at 55°C and 7 min at 55°C for the final extension. We used an iCycler thermal cycler (Bio-Rad Laboratories, USA). PCR products were fractionated in 2.5% agarose TBE gel. When the predicted PCR product sizes of the parental alleles differed by less than 5 bases, we used the GoTaq Colorless Master Mix (Promega) and a QIAxcel capillary electrophoresis system (Qiagen, USA).A linkage map was constructed using 175 SSR markers distributed among the 12 chromosomes (, , ) in the MAPMAKER/EXP 3.0 software (). The same 175 SSR markers were used to evaluate the isogenic status of the NILs. We performed QTL analysis for WBK and DTH by composite interval mapping in Windows QTL Cartographer 2.5 () as described in .To confirm detection of the QTLs, we analyzed homozygous plants from the 416 F2 population, which was derived from a cross between NIL2 and NW, for eight segregating SSR markers (RM1369 and RM8125 on chromosome 6; RM6971, RM2915, RM2482 and RM2255 on chromosome 9; and RM5352 and RM5494 on chromosome 10) as above. In addition, we used two-way analysis of variance (ANOVA) of the occurrence of WBK in the F2 population to identify interactions between QTLs. […]

Pipeline specifications

Software tools MAPMAKER, QTL Cartographer
Application WGS analysis
Organisms Oryza sativa