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 http://datingranking.net/cs/xdating-recenze/ 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 .
Data out-of parental genetic range
Hereditary variety anywhere between adult outlines are assessed having genome-large SNP markers because of the principal complement studies, team study, and also by good pairwise genome always check getting polymorphism between the moms and dads each and every people. To possess details, come across Most document 8.
Hereditary map construction
Genetic charts was created for each and every individual population just like the revealed before using CarthaGene entitled away from customized R texts. In the 1st step, mathematically strong scaffold charts was basically constructed with marker distances of on least ten cM. During the a moment step, ework charts containing as many indicators as you are able to, while maintaining a beneficial LOD score >step three.0 into the robustness regarding marker orders. Fundamentally, the whole charts was indeed received of the keeping more markers using bin-mapping . CentiMorgan (cM) ranges was indeed computed using Haldane’s mapping means . Private hereditary charts and genotypic study useful structure of the charts (Additional document 4) have been deposited in the MaizeGDB underneath the project phrase CORNFED .
Bodily chart coordinates regarding SNPs
Chromosome and you will status assignments from SNPs of MaizeSNP50 BeadChip offered by the manufacturer (Illumina ® , Hillcrest, Ca, USA), are based on the new B73 AGPv1 set-up with lots of indicators lacking an excellent chromosome and you will/or position recommendations. I for this reason performed an alternate mapping of one’s SNPs to the B73 AGPv2 assembly playing with BWA . The latest tasks were used for everyone analyses between the physical mapping information. Projects are available in Even more document cuatro.
Provided a great chromosome in addition to related hereditary chart of individuals population, we computed the fresh new marker ranks for the B73 set-up. From the real and you will genetic positions, we developed an initial Marey chart which has had every syntenic indicators. This Marey map try smoothed using cubic spline interpolations , producing an effective ‘bare’ Marey chart which was obligated to feel monotonic. Upcoming countries in which mapping recommendations was not having (including, segments IBD regarding the parents) had been disguised, generating ‘masked’ Marey maps (More file nine). The outlined process try told me inside the More document 8.