SNP genotyping
Bulk samples of dried leaves or kernels from up to eight D1 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 regarding adult hereditary variety
Genetic variety between adult traces try analyzed which have genome-broad SNP markers of the dominant enhance analysis, team research, by a good pairwise genome see to have polymorphism between the parents of each inhabitants. To own information, see Even more file 8.
Hereditary chart construction
Genetic maps was constructed each individual people just like the demonstrated earlier using CarthaGene titled from custom R texts. In the first action, mathematically powerful scaffold charts have been constructed with marker distances out-of on the very least ten cM. In the an additional step, ework maps who has as much indicators as you are able to, while maintaining a LOD score >step 3.0 toward robustness of marker orders. Eventually, the entire maps was basically acquired of the keeping extra markers using bin-mapping . CentiMorgan (cM) distances was indeed computed playing with Haldane’s mapping means . Private hereditary charts and genotypic studies used in construction of your maps (Even more document 4) was basically placed at MaizeGDB within the project acronym CORNFED .
Bodily chart coordinates from SNPs
Chromosome and you will position assignments of SNPs of the MaizeSNP50 BeadChip offered by the manufacturers. (Illumina ® , North park, California, USA), depend on the newest B73 AGPv1 construction with lots of indicators without an effective chromosome and/otherwise reputation guidance. I hence performed an alternate mapping of one’s SNPs toward B73 AGPv2 installation using BWA . The brand new assignments were utilized for everybody analyses between your actual mapping information. Projects appear in A lot more file 4.
Provided a great chromosome and sitio de citas de música gratis para solteros also the related hereditary map of people population, i computed the new marker ranking toward B73 installation. From these bodily and you will genetic ranks, we built a first Marey chart which includes most of the syntenic indicators. That it Marey chart is smoothed having fun with cubic spline interpolations , generating a ‘bare’ Marey map that has been compelled to getting monotonic. After that regions where mapping pointers is actually without having (like, locations IBD in the moms and dads) have been masked, producing ‘masked’ Marey charts (Additional document 9). The fresh new intricate procedure try informed me inside Extra file 8.