Color and pigment contents are important aspects of fruit quality and consumer acceptance of cucurbit crops. Here, we describe the independent mapping and cloning of a common causative APRR2 gene regulating pigment accumulation in melon and watermelon. We initially show that the APRR2 transcription factor is causative for the qualitative difference between dark and light green rind in both crops. Further analyses establish the link between sequence or expression level variations in the CmAPRR2 gene and pigment content in the rind and flesh of mature melon fruits. A genome-wide association study (GWAS) of young fruit rind color in a panel composed of 177 diverse melon accessions did not result in any significant association, leading to an earlier assumption that multiple genes are involved in shaping the overall phenotypic variation in this trait. Through resequencing of 25 representative accessions and allelism tests between light rind accessions, we show that multiple independent single nucleotide polymorphisms in the CmAPRR2 gene are causative of the light rind phenotype. The multi-haplotypic nature of this gene explains the lack of detection power obtained through genotyping by sequencing-based GWAS and confirms the pivotal role of this gene in shaping fruit color variation in melon. This study demonstrates the power of combining bi- and multi-allelic designs with deep sequencing, to resolve lack of power due to high haplotypic diversity and low allele frequencies. Due to its central role and broad effect on pigment accumulation in fruits, the APRR2 gene is an attractive target for carotenoid bio-fortification of cucurbit crops.

We have developed PotatoMASH (Potato Multi-Allele Scanning Haplotags), a novel low-cost, genome-scanning marker platform. We designed a panel of 339 multi-allelic regions placed at 1 Mb intervals throughout the euchromatic portion of the genome. These regions were assayed using a multiplex amplicon sequencing approach, which allows for genotyping hundreds of plants at a cost of 5 EUR/sample. We applied PotatoMASH to a population of over 700 potato lines. We obtained tetraploid dosage calls for 2012 short multi-allelic haplotypes in 334 loci, which ranged from 2 to 14 different haplotypes per locus. The system was able to diagnose the presence of targeted pest-resistance markers, to detect quantitative trait loci (QTLs) by genome-wide association studies (GWAS) in a tetraploid population, and to track variation in a diploid segregating population. PotatoMASH efficiently surveys genetic variation throughout the potato genome, and can be implemented as a single low-cost genotyping platform that will allow the routine and simultaneous application of marker-assisted selection (MAS) and other genotyping applications in commercial potato breeding programmes.

We genotyped a population of 618 diploid potato clones derived from six independent potato-breeding programmes from NW-Europe. The diploids were phenotyped for 23 traits, using standardized protocols and common check varieties, enabling us to derive whole population estimators for most traits. We subsequently performed a genome-wide association study (GWAS) to identify quantitative trait loci (QTL) for all traits with SNPs and short-read haplotypes derived from read-backed phasing. In this study, we used a marker platform called PotatoMASH (Potato Multi-Allele Scanning Haplotags); a pooled multiplex amplicon sequencing based approach. Through this method, neighboring SNPs within an amplicon can be combined to generate multiallelic short-read haplotypes (haplotags) that capture recombination history between the constituent SNPs and reflect the allelic diversity of a given locus in a different way than single bi-allelic SNPs. We found a total of 37 unique QTL across both marker types. A core of 10 QTL was detected with SNPs as well as with haplotags. Haplotags allowed to detect an additional 14 QTL not found based on the SNP set. Conversely, the bi-allelic SNP set also found 13 QTL not detectable using the haplotag set. We conclude that both marker types should routinely be used in parallel to maximize the QTL detection power. We report 19 novel QTL for nine traits: Skin Smoothness, Sprout Dormancy, Total Tuber Number, Tuber Length, Yield, Chipping Color, After-cooking Blackening, Cooking Type, and Eye depth.

Key message: Multiple QTLs control unreduced pollen production in potato. Two major-effect QTLs co-locate with mutant alleles of genes with homology to AtJAS, a known regulator of meiotic spindle orientation. In diploid potato the production of unreduced gametes with a diploid (2n) rather than a haploid (n) number of chromosomes has been widely reported. Besides their evolutionary important role in sexual polyploidisation, unreduced gametes also have a practical value for potato breeding as a bridge between diploid and tetraploid germplasm. Although early articles argued for a monogenic recessive inheritance, the genetic basis of unreduced pollen production in potato has remained elusive. Here, three diploid full-sib populations were genotyped with an amplicon sequencing approach and phenotyped for unreduced pollen production across two growing seasons. We identified two minor-effect and three major-effect QTLs regulating this trait. The two QTLs with the largest effect displayed a recessive inheritance and an additive interaction. Both QTLs co-localised with genes encoding for putative AtJAS homologs, a key regulator of meiosis II spindle orientation in Arabidopsis thaliana. The function of these candidate genes is consistent with the cytological phenotype of mis-oriented metaphase II plates observed in the parental clones. The alleles associated with elevated levels of unreduced pollen showed deleterious mutation events: an exonic transposon insert causing a premature stop, and an amino acid change within a highly conserved domain. Taken together, our findings shed light on the natural variation underlying unreduced pollen production in potato and will facilitate interploidy breeding by enabling marker-assisted selection for this trait.