SNP genotyping
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 wskazówki dotyczÄ…ce furfling 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 out-of adult hereditary range
Genetic diversity anywhere between adult contours is analyzed having genome-wider SNP markers by prominent accentuate research, party analysis, and by good pairwise genome check always to possess polymorphism between your moms and dads of each population. To possess information, see More file 8.
Hereditary map build
Genetic charts had been constructed per private society since revealed prior to having fun with CarthaGene titled regarding custom R programs. In the 1st action, mathematically strong scaffold maps was built with marker ranges of in the minimum 10 cM. In a second action, ework maps which has as numerous indicators that you could, while keeping good LOD get >step 3.0 toward robustness of marker instructions. In the end, the complete charts was gotten by the keeping of additional markers using bin-mapping . CentiMorgan (cM) ranges had been computed playing with Haldane’s mapping function . Individual genetic charts and you will genotypic studies utilized for structure of your own charts (More file 4) was basically deposited within MaizeGDB within the enterprise acronym CORNFED .
Actual chart coordinates out-of SNPs
Chromosome and you can standing assignments of SNPs of your own MaizeSNP50 BeadChip given by the product manufacturer (Illumina ® , North park, California, USA), depend on new B73 AGPv1 assembly with quite a few markers lacking an effective chromosome and you will/otherwise status information. I thus did a different mapping of your SNPs into the B73 AGPv2 assembly having fun with BWA . The fresh assignments were utilized for everyone analyses involving the bodily mapping suggestions. Projects come in Even more file 4.
Considering a beneficial chromosome therefore the associated hereditary chart of an individual population, i computed the new marker positions with the B73 set-up. From the actual and you will hereditary ranks, i developed a primary Marey map which includes every syntenic indicators. Which Marey map are smoothed using cubic spline interpolations , producing good ‘bare’ Marey chart which was compelled to end up being monotonic. Upcoming regions where mapping suggestions try devoid of (such, places IBD on the mothers) was basically disguised, generating ‘masked’ Marey maps (Additional file 9). New in depth process try explained for the More file 8.