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 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 .
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Analysis regarding adult hereditary variety
Genetic range anywhere between adult outlines is reviewed having genome-wider SNP markers from the dominant enhance data, class investigation, and by a great pairwise genome examine to own polymorphism between your parents of every population. Getting details, get a hold of A lot more document 8.
Genetic chart construction
Genetic charts was basically developed each personal people just like the revealed earlier playing with CarthaGene entitled regarding custom Roentgen scripts. In the first action, statistically powerful scaffold maps were built with marker distances of in the minimum ten cM. Inside the another action, ework charts who has as many indicators you could, while maintaining a good LOD rating >step 3.0 to the robustness away from marker commands. Fundamentally, the entire charts was obtained because of the keeping of more indicators using bin-mapping . CentiMorgan (cM) distances have been calculated playing with Haldane’s mapping setting . Personal hereditary charts and you can genotypic analysis useful for design of charts (Extra document 4) was in fact deposited during the MaizeGDB under the enterprise acronym CORNFED .
Bodily map coordinates out of SNPs
Chromosome and you can updates projects out-of SNPs of the MaizeSNP50 BeadChip provided by the manufacturers. (Illumina ® , Hillcrest, California, USA), depend on brand new B73 AGPv1 set up with several indicators without having an effective chromosome and/otherwise condition guidance. I hence performed another mapping of the SNPs towards the B73 AGPv2 installation having fun with BWA . New tasks were used for all analyses within bodily mapping advice. Tasks are available in More document cuatro.
Offered an excellent chromosome and the related hereditary chart of an individual people, we calculated the latest marker ranking on B73 set-up. Because of these physical and you will hereditary ranks, we created an initial Marey chart containing the syntenic markers. That it Marey map was smoothed using cubic spline interpolations , creating an effective ‘bare’ Marey chart which was obligated to feel monotonic. Next places where mapping advice was without (like, segments IBD from the mothers) was in fact disguised, generating ‘masked’ Marey maps (Extra file nine). The fresh intricate processes are said in Even more file 8.