SNP genotyping
Bulk samples of dried leaves or kernels from up to eight Dstep step 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 .
Research out of adult hereditary diversity
Hereditary diversity ranging from parental contours was analyzed which have genome-large SNP indicators from the dominating complement research, cluster investigation, and by a beneficial pairwise genome examine to possess polymorphism within parents of each inhabitants. For facts, look for Most file 8.
Genetic map framework
Genetic charts were constructed for each and every individual populace due to the fact discussed before playing with CarthaGene called away from customized R programs. In the first step, mathematically robust scaffold charts were built with marker distances of in the least ten cM. Inside the a second step, ework charts who has as many indicators as you are able to, while keeping a beneficial LOD get >3.0 with the robustness of marker commands. Fundamentally, the entire charts was acquired because of the placement of extra indicators having fun with bin-mapping . CentiMorgan (cM) distances was indeed calculated playing with Haldane’s mapping form . Individual genetic charts and you can genotypic study useful for design of your own maps (Additional document 4) was basically transferred during the MaizeGDB according to the enterprise phrase CORNFED .
Physical map coordinates from SNPs
Chromosome and you will updates projects out of SNPs of your MaizeSNP50 BeadChip supplied by the manufacturer (Illumina ® , North park, California, USA), are based on this new B73 AGPv1 system with many indicators lacking an effective chromosome and you will/or reputation guidance. I thus did yet another mapping of your own SNPs towards B73 AGPv2 assembly having fun with BWA . This new tasks were used for everyone analyses within actual mapping advice. Projects can be found in More file 4.
Given a beneficial chromosome additionally the related hereditary map of men and women society, i calculated the new marker positions with the B73 set-up. From the real and hereditary ranks, we created a first Marey map which includes all the syntenic indicators. So it Marey map is actually smoothed playing with cubic spline interpolations https://datingranking.net/pl/chatib-recenzja/, creating a ’bare’ Marey chart that has been forced to become monotonic. Upcoming regions where mapping recommendations was not having (for example, places IBD throughout the parents) have been disguised, promoting ’masked’ Marey charts (Extra file nine). The outlined process are explained within the A lot more file 8.
