Bulk samples of dried leaves or kernels from up to eight Dstep step one plants derived from the same D0, were used for DNA extraction using the cetyl trimethylammonium bromide (CTAB) procedure. DNA samples were adjusted to 50 to 70 ng/?l and 200 ng per sample were used for genotyping. DH line purity and integrity was first checked using a custom 96plex VeraCode assay (Illumina ® , San Diego, CA, USA) with genome-wide SNP markers to ensure that the lines carried only one of the parental alleles at each SNP, that they did not carry alleles of the inducer line and that they were derived from true F1 plants. For a subset of DH lines, 13 proprietary SNP markers assayed with the KASP™ technology (LGC Genomics, Berlin, Germany) were used for testing line purity and integrity. True DH lines were then used for genotyping with the Illumina ® MaizeSNP50 BeadChip on an Illumina ® iScan platform. Array hybridization and raw data processing were performed according to manufacturer’s instructions (Illumina ® ). Raw data were analyzed in Illumina ® ‘s Genome Studio software version v2011 (Illumina ® ) using an improved version of the public cluster file (MaizeSNP50_B.egt, ). SNP data were filtered based on the GTscore using a threshold of 0.7. Heterozygous SNPs were set to missing values (NA) and only markers with a minor allele frequency >0.1 per population were used for mapping. For each population, the allele of the central line was coded as the ‘A’ allele, and the allele of the founder line was coded as ‘B’ allele (Additional file 4). Raw genotyping data of parents and DH lines are available at NCBI Gene Expression Omnibus as dataset GSE50558 .
Data off parental hereditary variety
Genetic assortment between parental lines is actually analyzed which have genome-large SNP markers because of the dominating accentuate study, group data, by a pairwise genome search to have polymorphism involving the mothers each and podÅ‚Ä…czenie mytranssexualdate every population. To possess info, come across Even more file 8.
Hereditary chart design
Hereditary charts were built for each and every private people due to the fact explained before using CarthaGene named from customized Roentgen scripts. In the 1st action, statistically robust scaffold maps was indeed designed with marker distances out-of from the the very least ten cM. Within the the second step, ework charts with as much indicators as you are able to, while maintaining a good LOD rating >step three.0 on the robustness from marker instructions. Eventually, the complete charts had been received of the placement of more indicators playing with bin-mapping . CentiMorgan (cM) ranges had been determined playing with Haldane’s mapping mode . Personal genetic maps and you can genotypic study used in build of your own charts (Extra document cuatro) was indeed transferred at the MaizeGDB according to the opportunity acronym CORNFED .
Physical map coordinates away from SNPs
Chromosome and position assignments from SNPs of your MaizeSNP50 BeadChip supplied by the manufacturers. (Illumina ® , San diego, Ca, USA), depend on the fresh B73 AGPv1 construction with lots of indicators lacking good chromosome and you can/otherwise position information. I for this reason performed another mapping of your own SNPs on the B73 AGPv2 system having fun with BWA . The fresh tasks were utilized for everybody analyses within actual mapping guidance. Tasks are available in Even more file cuatro.
Given good chromosome additionally the relevant genetic chart of men and women populace, we calculated the latest marker positions towards the B73 installation. From these physical and genetic ranking, we constructed a first Marey chart that has had every syntenic indicators. It Marey chart are smoothed having fun with cubic spline interpolations , promoting a ‘bare’ Marey map which had been obligated to getting monotonic. After that places in which mapping recommendations try not having (such as for instance, areas IBD in the mothers) was basically disguised, generating ‘masked’ Marey charts (Most document 9). This new outlined techniques was informed me when you look at the Extra document 8.