Genetic Linkage Map Construction and Genetic Mapping of the Pigmentation Gene

Only co-dominant markers were used for the linkage map construction. The segregation of alleles at the SSR or SNP marker loci was checked against the expected ratios for codominant (1:2:1) markers using a chi-square test. The genetic linkage map was constructed with MAPMAKER/EXP (version 3.0b) (Lincoln et al., 1993) using genotyping data from polymorphic and co-dominant markers from the 298 SSR and 1536 SNP marker sets. Map distances in centiMorgan (cM) were converted from recombination fractions using the Kosambi mapping function. Two-point analysis was used to identify linkage groups (LGs) with an LOD score of 6.0 and a maximum distance of 40 cM, except for LGs 13 and 15, in which specific markers were grouped with a LOD value of 3.5. Three-point and multi-point analyses were used to determine the order and interval distances between the markers in each LG. Loci whose position were ambiguous (i.e., those placed automatically at a less-strict LOD of 2.0) were noted. Markers that had the most skewed segregation ratios (P < 0.0001) were excluded from the map. Linkage maps were drawn using MapChart 2.1 software (Voorrips, 2002). The linkage groups were randomly numbered as there are no previously reported O. cumana genetic maps. Simple correlation coefficients (r) between the total number of markers per linkage group and the total linkage group length were calculated. The significance of the correlation coefficients was calculated by the standard testing procedures for r = 0 null hypothesis (Snedecor and Cochran, 1989).

As the “plant pigmentation” trait is controlled by a single gene (P_g, Rodríguez-Ojeda et al., 2011), it was mapped as a Mendelian locus. Accordingly, the genotypes for the P_g gene were inferred from the pigmentation phenotypes in F₂ plants and their corresponding F_2:3 families. F₂ plants were classified as homozygous dominant for the pigmentation gene if they showed a stem similar to EK-12, and showed uniformly pigmented plants in their respective F₃ progeny, heterozygous if their F₃ segregated for stem color, and homozygous recessive if they were similar to EK-A1 and showed uniformly unpigmented plants in their respective F₃ progeny. Linkage analysis for the pigmentation gene was run with MAPMAKER/EXP (version 3.0b) using segregation data for SNP and SSR marker loci and for the P_g locus. P_g mapping was carried out as indicated for SSR and SNP markers, excepting that a LOD threshold of 10 and a maximum distance of 30 cM were used as linkage criteria. Finally, potential candidate genes for the Pg locus were identified using BLAST searches for plant pigment biosynthesis genes at the flavonoid/anthocyanin and carotenoid biosynthesis pathways (MetaCyc, version 20.0; Caspi et al., 2018) against a 622 contigs first draft of the O. cumana sequence genome (Gouzy et al., 2017).