Bulk samples of dried leaves or kernels from up to eight Dstep 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 .
Investigation out of parental hereditary variety
Hereditary range anywhere between parental lines are analyzed with genome-wide SNP markers by principal accentuate investigation, group investigation, and by an effective pairwise genome test for polymorphism within moms and dads of each population. Getting information, discover Additional document 8.
Hereditary map design
Hereditary maps was indeed constructed for each individual society because the revealed earlier using CarthaGene called from custom Roentgen scripts. In the 1st step, statistically robust scaffold maps was constructed with marker distances away from on least 10 cM. During the one minute action, ework charts which has had as numerous indicators as possible, while maintaining a great LOD score >3.0 towards the robustness from marker requests. In the end, the complete charts was indeed received from the placement of additional indicators having fun with bin-mapping . CentiMorgan (cM) distances had been determined having fun with Haldane’s mapping means . Individual genetic charts and you can genotypic analysis utilized for design of one’s maps (Additional file cuatro) was basically placed at the MaizeGDB within the investment acronym CORNFED .
Actual chart coordinates out of SNPs
Chromosome and you can status assignments from SNPs of one’s MaizeSNP50 BeadChip given by the product manufacturer (Illumina ® , North park, Ca, USA), are derived from the fresh B73 AGPv1 construction with many different indicators devoid of a chromosome and/otherwise standing pointers. We thus performed an alternate mapping of one’s SNPs towards B73 AGPv2 system having fun with BWA . The new tasks were utilized for all analyses involving the real mapping guidance. Assignments are available in Even more file cuatro.
Offered a chromosome in addition to related hereditary chart of individuals population, i calculated the fresh marker positions on B73 assembly. From the actual and you will genetic positions, we constructed an initial Marey chart that contains the syntenic markers. This Marey chart is actually smoothed using cubic spline interpolations , producing a good ‘bare’ Marey map that has been obligated to be monotonic. Up coming countries where mapping information is actually without having (particularly, avenues IBD regarding the mothers) had been disguised, producing ‘masked’ Marey maps (Extra document nine). The fresh new in depth process are informed me when you look at the A lot more document 8.