Bulk samples of dried leaves or kernels from up to eight Dstep 1 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 .
Studies out of adult hereditary range
Hereditary diversity ranging from parental lines try assessed which have genome-wider SNP markers by the principal enhance studies, class research, and also by an excellent pairwise genome examine getting polymorphism between the moms and dads of each and every population. For info, look for Extra file 8.
Genetic chart structure
Genetic maps was in fact developed for each and every private society as the described prior to using CarthaGene named out of custom R texts. In the 1st action, mathematically sturdy scaffold maps was basically designed with marker distances regarding within least ten cM. In an additional action, ework maps that features as numerous indicators that one can, while keeping a good LOD get >step 3.0 to your robustness out of marker instructions. Ultimately, the whole charts was indeed obtained because of the placement of even more indicators having fun with bin-mapping . CentiMorgan (cM) ranges was determined using Haldane’s mapping setting . Private hereditary maps and you may genotypic study useful structure of charts (Additional file 4) was indeed placed on MaizeGDB according to the endeavor phrase CORNFED .
Real chart coordinates off SNPs
Chromosome and you will standing tasks out-of SNPs of the MaizeSNP50 BeadChip given by the product manufacturer (Illumina ® , Hillcrest, Ca, USA), derive from the fresh new B73 AGPv1 system with lots of markers not having an excellent chromosome and you may/otherwise updates advice. We hence performed a new mapping of the SNPs to the B73 AGPv2 assembly using BWA . New projects were utilized for everybody analyses between your real mapping suggestions. Tasks can be found in Even more document cuatro.
Considering a chromosome additionally the relevant hereditary map of men and women people, we determined new marker positions to the B73 system. Because of these physical and you can hereditary ranking, we created an initial Marey chart which has most of the syntenic indicators. It Marey map was smoothed playing with cubic spline interpolations , generating a beneficial ‚bare‘ Marey chart that has been forced to be monotonic. Then nations in which mapping suggestions try without having (such as for example, locations IBD on mothers) was basically masked, promoting ‚masked‘ Marey charts (Most document 9). The fresh detail by detail procedure was said in the Even more document 8.