Identification of alleles of carotenoid pathway genes important for zeaxanthin accumulation in potato tubers

We have investigated the genetics and molecular biology of orange flesh colour in potato (Solanum tuberosum L.). To this end the natural diversity in three genes of the carotenoid pathway was assessed by SNP analyses. Association analysis was performed between SNP haplotypes and flesh colour phenotypes in diploid and tetraploid potato genotypes. We observed that among eleven beta-carotene hydroxylase 2 (Chy2) alleles only one dominant allele has a major effect, changing white into yellow flesh colour. In contrast, none of the lycopene epsilon cyclase (Lcye) alleles seemed to have a large effect on flesh colour. Analysis of zeaxanthin epoxidase (Zep) alleles showed that all (diploid) genotypes with orange tuber flesh were homozygous for one specific Zep allele. This Zep allele showed a reduced level of expression. The complete genomic sequence of the recessive Zep allele, including the promoter, was determined, and compared with the sequence of other Zep alleles. The most striking difference was the presence of a non-LTR retrotransposon sequence in intron 1 of the recessive Zep allele, which was absent in all other Zep alleles investigated. We hypothesise that the presence of this large sequence in intron 1 caused the lower expression level, resulting in reduced Zep activity and accumulation of zeaxanthin. Only genotypes combining presence of the dominant Chy2 allele with homozygosity for the recessive Zep allele produced orange-fleshed tubers that accumulated large amounts of zeaxanthin

A highly robust and optimized sequence-based approach for genetic polymorphism discovery and genotyping in large plant populations

Advent of the next generation sequencing techniques motivates recent interest in developing sequence-based identification and genotyping of genome-wide genetic variants in large populations, with RAD-seq being a typical example. Without taking proper account for the fact that chloroplast and rRNA genes may occupy up to 60 % of the resulting sequence reads, the current RAD-seq design could be very inefficient for plant and crop species. We presented here a generic computational tool to optimize RAD-seq design in any plant species and experimentally tested the optimized design by implementing it to screen for and genotype sequence variants in four plant populations of diploid and autotetraploid Arabidopsis and potato Solanum tuberosum. Sequence data from the optimized RAD-seq experiments shows that the undesirable chloroplast and rRNA contributed sequence reads can be controlled at 3–10 %. Additionally, the optimized RAD-seq method enables pre-design of the required uniformity and density in coverage of the high quality sequence polymorphic markers over the genome of interest and genotyping of large plant or crop populations at a competitive cost in comparison to other mainstream rivals in the literature