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, https://datingranking.net/pl/fdating-recenzja/ ). 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 out of adult genetic diversity
Genetic assortment ranging from parental outlines is examined that have genome-broad SNP indicators by the dominant complement investigation, people studies, and also by good pairwise genome check always to own polymorphism amongst the moms and dads of every people. Having information, pick Most document 8.
Genetic map design
Hereditary maps were created for each and every private inhabitants given that revealed earlier playing with CarthaGene named off custom R programs. In the first step, statistically powerful scaffold maps was basically built with marker ranges regarding from the least ten cM. During the the second step, ework charts containing as much markers that one can, while maintaining a good LOD score >step 3.0 towards the robustness from marker commands. Ultimately, the entire charts was in fact obtained of the keeping of even more indicators using bin-mapping . CentiMorgan (cM) ranges was indeed calculated having fun with Haldane’s mapping function . Individual genetic maps and you may genotypic investigation used for construction of maps (Additional file 4) was basically deposited on MaizeGDB under the enterprise phrase CORNFED .
Actual map coordinates of SNPs
Chromosome and you may reputation assignments out of SNPs of the MaizeSNP50 BeadChip offered by the manufacturers. (Illumina ® , San diego, Ca, USA), derive from the brand new B73 AGPv1 assembly with quite a few indicators without a chromosome and you will/otherwise updates recommendations. I for this reason did a unique mapping of one’s SNPs to your B73 AGPv2 system using BWA . The assignments were used for everybody analyses between the real mapping pointers. Projects come in Extra document cuatro.
Provided good chromosome and the relevant genetic chart of an individual populace, we determined the newest marker positions toward B73 construction. From these real and you can hereditary positions, i created a first Marey map containing all of the syntenic indicators. This Marey chart are smoothed using cubic spline interpolations , creating an effective ‘bare’ Marey map that has been obligated to getting monotonic. Up coming places where mapping information try not having (such, locations IBD about parents) was basically masked, generating ‘masked’ Marey charts (More document nine). This new detail by detail procedure is actually said during the Extra document 8.