SNP genotyping

Bulk samples of dried leaves or kernels from up to eight D_{step step 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 .

Study out of parental hereditary variety

Hereditary range anywhere between adult traces try analyzed having genome-wide SNP markers from the prominent coordinate research, party study, by an effective pairwise genome check for polymorphism amongst the mothers of each and every populace. Getting details, pick More document 8.

Genetic map framework

Genetic maps was basically developed for every single individual population since described before playing with CarthaGene named away from customized R texts. In the first action, statistically strong scaffold charts was designed with marker ranges of at the very least ten cM. When you look at the one minute step, ework maps which has had as many markers you could, while maintaining good LOD rating >step 3.0 toward robustness of marker commands. Eventually, the complete charts were obtained by the keeping of even more indicators playing with bin-mapping . CentiMorgan (cM) distances had been computed using Haldane’s mapping mode . Personal genetic charts and you may genotypic study useful for structure of your charts (Additional file 4) was placed in the MaizeGDB according to the enterprise phrase CORNFED .

Bodily map coordinates regarding SNPs

Chromosome and you can condition tasks of SNPs of one’s MaizeSNP50 BeadChip supplied by the product manufacturer (Illumina ® , North park, Ca, USA), are derived from the fresh B73 AGPv1 system with lots of markers lacking good chromosome and you can/or reputation guidance. We hence performed a special mapping of one’s SNPs towards B73 AGPv2 set-up having fun with BWA . The brand new projects were used for everybody analyses between your bodily mapping advice. Tasks can be found in More file cuatro.

Considering a great chromosome and related genetic map of an individual people, we determined the fresh marker positions towards the B73 set up. From these real and you will genetic ranks, we created an initial Marey chart which includes most of the syntenic indicators. It Marey chart is actually smoothed having fun with cubic spline interpolations , promoting a good ‘bare’ Marey chart that was forced to become monotonic. Upcoming places in which mapping pointers try without having (such as for instance, markets IBD in the moms and dads) have been disguised, producing ‘masked’ Marey charts (A lot more kody promocyjne aisle document nine). The latest intricate procedure try explained in Additional file 8.