SNP genotyping

Bulk samples of dried leaves or kernels from up to eight D_{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) przykÅ‚ady profili bookofsex 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 parental hereditary diversity

Hereditary variety ranging from parental outlines try assessed with genome-broad SNP indicators from the dominating coordinate study, group data, and by good pairwise genome inspect to possess polymorphism amongst the moms and dads of each and every people. Having details, find Extra file 8.

Hereditary chart design

Hereditary charts had been built for each individual people because described before having fun with CarthaGene titled off individualized R programs. In the first step, statistically strong scaffold maps have been built with marker ranges from from the minimum ten cM. For the an additional step, ework maps which includes as numerous indicators as you are able to, while maintaining a good LOD get >3.0 on the robustness out of marker orders. In the end, the complete charts was in fact obtained by the keeping additional indicators playing with bin-mapping . CentiMorgan (cM) distances was in fact calculated using Haldane’s mapping function . Private hereditary maps and you can genotypic data useful build of maps (Most document cuatro) had been deposited within MaizeGDB within the venture phrase CORNFED .

Bodily chart coordinates regarding SNPs

Chromosome and you can standing projects out of SNPs of your MaizeSNP50 BeadChip provided by the manufacturers. (Illumina ® , Hillcrest, Ca, USA), are derived from the newest B73 AGPv1 installation with many markers devoid of a good chromosome and/or position recommendations. We therefore performed an alternative mapping of your own SNPs to your B73 AGPv2 set-up playing with BWA . The fresh new assignments were used for all analyses between the actual mapping recommendations. Assignments come in Additional document 4.

Given a great chromosome together with relevant hereditary chart of an individual people, we determined the brand new marker ranking for the B73 assembly. Because of these real and genetic ranking, we built a primary Marey map which has had every syntenic markers. Which Marey chart is actually smoothed using cubic spline interpolations , generating an effective ‘bare’ Marey map that was obligated to getting monotonic. Following nations in which mapping advice is actually without having (including, areas IBD on the moms and dads) was indeed disguised, generating ‘masked’ Marey maps (Additional file nine). The new intricate procedure is told me when you look at the A lot more document 8.