SNP genotyping
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, ). 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 from parental genetic assortment
Hereditary assortment between parental contours try examined having genome-large SNP indicators by dominant coordinate study, group studies, and also by good pairwise genome check getting polymorphism between your mothers of any populace. To possess info, get a hold of A lot more file 8.
Genetic map framework
Genetic maps was built each personal people as explained prior to playing with CarthaGene called out-of personalized Roentgen scripts. In the 1st action, statistically powerful scaffold charts were constructed with marker ranges out of on the very least ten cM. When you look at the a second step, ework maps with as many indicators that you could, while keeping a good LOD get >step three.0 to your robustness away from marker commands. Eventually, the complete charts have been gotten by the keeping a lot more markers having fun with bin-mapping . CentiMorgan (cM) ranges have been calculated playing with Haldane’s mapping setting . Private hereditary maps and you will genotypic studies used for framework of your own charts (Most file 4) had been placed in the MaizeGDB beneath the investment phrase CORNFED .
Bodily map coordinates from SNPs
Chromosome and condition projects out-of SNPs of one’s MaizeSNP50 BeadChip given by the manufacturers. (Illumina ® , San diego, Ca, USA), depend on this new B73 AGPv1 assembly with many different markers kupony jackd without having a good chromosome and you can/otherwise standing pointers. I therefore performed an alternate mapping of the SNPs towards B73 AGPv2 construction playing with BWA . Brand new assignments were utilized for all analyses within physical mapping advice. Projects appear in A lot more file cuatro.
Provided a chromosome and also the associated hereditary chart of men and women inhabitants, i determined the newest marker positions towards B73 set up. From these actual and hereditary ranking, we built a primary Marey map that features all syntenic indicators. That it Marey map is smoothed playing with cubic spline interpolations , creating good ‘bare’ Marey map that was obligated to become monotonic. Next nations where mapping pointers was without having (such as for example, segments IBD on the mothers) were masked, creating ‘masked’ Marey maps (Extra file nine). The fresh new detailed techniques is actually said from inside the Most document 8.
