Computational protocol: High-resolution QTL mapping for grain appearance traits and co-localization of chalkiness-associated differentially expressed candidate genes in rice

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Protocol publication

[…] Images of the mature grain were captured on a CanoScan 5600 F (Canon, Japan) scanner with the supplied software without image enhancement, and the grain shape parameters of GW, GL, LWR, CS, PL, and AS were measured precisely using SmartGrain Software (Tanabata et al. ). The chalkiness parameters were measured with an automatic machine JMWT 12 according to Xu et al. (). Two metrics were used to describe grain chalkiness as previously described (Zhao et al. ): percentage of grains with chalk (PGWC) and degree of endosperm chalkiness (DEC) which is the ratio of total chalky area to the total kernel area of all sampled grains. The statistical analysis was performed with SPSS statistics 18.0 and Microsoft Excel. [...] Leaf samples were collected from two parental lines and 192 RILs at F7 generation. DNAs were extracted using the CTAB method and quantified using both a NanoDrop ND-1000 Spectrophotometer and agarose gel electrophoresis. In our study, the genome of parental lines, PYZX and P02428, were directly sequenced to ~25× coverage, while the RILs were subjected to Genotyping By Sequencing (GBS) as described by Elshire et al. (). The DNA samples of the RIL population digested using MseI and HaeIII. The other basic schematic of the protocol used for performing GBS was according to Duan et al. ().Sequencing was performed on the Illumina HiSeq 2500 platform to generate 150 bp paired-end reads (Novogene Bioinformatics Technology Co., Ltd, China). The original image data generated were converted into sequence data via base calling (Illumina pipeline CASAVA v1.8.2) and then subjected to the quality control (QC) procedure to remove unusable reads: 1) reads contain the Illumina library construction adapters; 2) reads contain more than 10 % unknown bases (N bases); 3) one end of a read contains more than 50 % low quality bases 4) Sequencing reads were aligned to the reference genome (http://plants.ensembl.org/Oryza_sativa/) using BWA with default parameters. Subsequent processing, including duplicate removal was performed using SAMtools and PICARD (http://picard.sourceforge.net). The raw SNP/InDel sets are called by SAMtools with the parameters as ‘-q 1 -C 50 –m 2 -F 0.002 -d 1000’. Then we filtered these sets using the following criteria: (1) mapping quality >20; (2) depth of the variant position >4. […]

Pipeline specifications

Software tools SPSS, BaseSpace, BWA, SAMtools, Picard
Applications Miscellaneous, GBS analysis
Diseases Angelman Syndrome