2016 85036 NG

To independently verify the order of superscaffolds along the nine pseudomolecules, F₂ population 85036, consisting of 84 individuals (unpublished data), was used to generate a linkage map including GBS SNP markers. Sequencing and library preparation was carried out at the Biotechnology Center, UW-Madison (WI, USA). DNA was quantified using Quantus PicoGreen ds DNA Kit (Life Technologies, Grand Island, NY) and normalized to 10ng/μl. 50 ng of DNA was used for each GBS reaction. GBS libraries were prepared as described by Elshire et al., with minimal modification and half-sized reactions.

The resulting reads were analyzed using TASSEL version 4.3.11 with paired-end data preprocessed for TASSEL compatibility with bb.tassel (https://github.com/dsenalik/bb). SNPs were called using documented GBS pipeline procedures, with non-default parameter of mintagoccurrence=2. A total of 85,178 SNPs were obtained. Only sequences containing SNPs that unambiguously aligned to the carrot genome assembly were kept (18,007 SNPs). Finally, SNPs scored as heterozygous but with an allele ratio A:B far from 1:1 were eliminated if the ratio was < 0.3 or > 3.0, where A and B were the two alleles for a given SNP, leaving 516 high quality markers for linkage mapping.

Mapping was carried out with JoinMap 4.0. Population-specific locus genotype scores were then integrated into one dataset in each Linkage Group (LG) using the Combine Groups for Mapping Integration Module, followed by locus ordering by the Regression Mapping Module of JoinMap. The following parameters were used for the calculation: Kosambi's mapping function, LOD ≥ 3.0, REC frequency ≤0.4, goodness of fit jump threshold for removal of loci = 5.0, number of added loci after which a ripple is performed = 1, and third round = yes. Markers in common were used as anchor points. The order of markers across the linkage map was verified using CheckMatrix (http://www.atgc.org/XLinkage). Markers that were inconsistently placed due to false double recombination events were removed. The resulting map covered 450 cM and included 394 markers. At LOD > 10, with less than 10% missing data for marker and genotype, 394 markers were grouped into nine linkage groups.