16.marker.63

Inheritance of purple pigmentation was studied in F₂, F₃ and F₄ families derived from an initial cross between P4201 [58262⊗] and B6320 [96'606-4]. P4201 is an inbred line with purple outer phloem and yellow xylem storage roots and purple leaves that was derived from a cross between inbred P9547 with purple xylem and phloem root color derived from Central Anatolia, and B2566, an inbred with orange root color from diverse European sources. B6320 is an inbred with orange roots and green petioles derived from the European open-pollinated cultivars Nantes and Camberley. A single F₁ plant with purple root outer phloem and yellow xylem, and purple leaves, was self-pollinated to produce the F₂ population 70349 (N = 519), which was used for genetic mapping studies.

Total genomic DNA of individual plants from 70349, 2170 and 10117 populations was isolated from lyophilized leaves following the protocol described by Murray and Thompson and quantified using Pico Green (Invitrogen, Paisley, UK).

The linkage map was constructed using 187 F₂ individuals from 70349. A collection of 4000 published SNPs developed from carrot transcriptome data and 40 published SSR markers with known chromosome location were used. SNPs were genotyped using the KASPar chemistry, which is a competitive allele-specific PCR SNP genotyping system using FRET quencher cassette oligos (http://www.lgcgroup.com). SNPs were coded with a ‘K’ followed by four digit numbers (e.g. K0001). SNPs located within carotenoid or anthocyanin biosynthetic genes were labeled according to the gene abbreviation. SSR primer pairs were evaluated using a fluorescent method as described before.

JoinMap 4.0 software was used for mapping. Scores of all markers used for mapping were converted into genotype codes using the A/H/B system for co-dominant and A/C, B/D system for dominant markers segregating in F₂ population. The linkage groups (LGs) were obtained at a LOD threshold value >3.0. Regression mapping algorithm and Haldane’s mapping function was used to calculate genetic distances among marker loci. Markers and genotypes with more than 10% of missing data were excluded from the analysis. The degree of marker segregation distortion in the F₂ was determined by marker data comparison against the expected 3:1 and 1:2:1 ratio for dominant and codominant markers, respectively, using Chi square tests, where significant distortion was declared at P < 0.01. The marker order in each linkage group was examined for inconsistencies leading to false double recombination events using CheckMatrix (http://www.atgc.org/XLinkage). Markers with more than one inconsistent score were removed. In order to reduce the complexity of the final map figure, redundant markers, considering as such those that had no recombination among them and therefore shared the same map position, were removed from the image, leaving a single marker -the marker with the least amount of missing data- per map position.