SNP genotyping
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 .
Research from adult hereditary range
Hereditary assortment ranging from parental outlines is actually reviewed with genome-greater SNP markers of the dominant complement analysis, class studies, and also by an excellent pairwise genome see for polymorphism between your mothers each and every society. Having information, get a hold of Additional document 8.
Hereditary map construction
Hereditary maps have been built for each individual populace as the discussed earlier having fun with CarthaGene titled out of personalized Roentgen scripts. In the 1st action, statistically sturdy scaffold charts was basically built with marker ranges regarding on minimum ten cM. Into the an extra action, ework maps that has had as numerous markers that one can, while maintaining a LOD get >step 3.0 toward robustness away from marker instructions. In the long run, the whole maps was indeed acquired because of the keeping extra markers having fun with bin-mapping . CentiMorgan (cM) ranges was in fact calculated playing with Haldane’s mapping means . Individual genetic charts and you will genotypic investigation used for design of your own charts (A lot more file cuatro) had been deposited on MaizeGDB according to the venture acronym CORNFED .
Real chart coordinates from SNPs
Chromosome and status tasks regarding SNPs of your MaizeSNP50 BeadChip offered by the manufacturers. (Illumina ® , San diego, California, USA), are derived from the B73 AGPv1 set up with lots of markers lacking a great chromosome and you will/otherwise standing pointers. We ergo performed a unique mapping of SNPs into the B73 AGPv2 system playing with BWA . This new projects were utilized for everybody analyses amongst the actual mapping suggestions. Assignments come in Extra file cuatro.
Considering good chromosome additionally the associated genetic map of an individual society, i computed this new marker ranks for the B73 system. From the physical and you may genetic positions, we built an initial Marey map that contains all of the syntenic markers. So it Marey chart are smoothed having fun with cubic spline interpolations , promoting an effective ‘bare’ Marey chart that was compelled to be monotonic. Next nations where mapping recommendations are lacking (such as for example, avenues IBD throughout the moms and dads) was disguised, creating ‘masked’ Marey charts (Extra file nine). The fresh new detailed procedure is informed me inside the Even more document 8.