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.

SUMMARYIn the development of tomato compound leaves, local auxin maxima points, separated by the expression of the Aux/IAA protein SlIAA9/ENTIRE (E), direct the formation of discrete leaflets along the leaf margin. The local auxin maxima promote leaflet initiation, while E acts between leaflets to inhibit auxin response and lamina growth, enabling leaflet separation. Here, we show that a group of auxin response factors (ARFs), which are targeted by miR160, antagonizes auxin response and lamina growth in conjunction with E. In wild-type leaf primordia, the miR160-targeted ARFs SlARF10A and SlARF17 are expressed in leaflets, and SlmiR160 is expressed in provascular tissues. Leaf overexpression of the miR160-targeted ARFs SlARF10A, SlARF10B or SlARF17, led to reduced lamina and increased leaf complexity, and suppressed auxin response in young leaves. In agreement, leaf overexpression of miR160 resulted in simplified leaves due to ectopic lamina growth between leaflets, reminiscent of e leaves. Genetic interactions suggest that E and miR160-targeted ARFs act partially redundantly but are both required for local inhibition of lamina growth between initiating leaflets. These results show that different types of auxin signal antagonists act cooperatively to ensure leaflet separation in tomato leaf margins.

23Color and pigment content are important aspects of fruit quality and consumer 24 acceptance of cucurbit crops. Here, we describe the independent mapping and cloning 25 of a common causative APRR2 gene regulating pigment accumulation in melon and 26 watermelon. We initially show that the APRR2 transcription factor is causative for the 27 qualitative difference between dark and light green rind in both crops. Further analyses 28 establish the link between sequence or expression level variations in the CmAPRR2 29 gene and pigments content in the rind and flesh of mature melon fruits. GWAS of young 30 fruit rind color in a panel composed of 177 diverse melon accessions did not result in 31 any significant association, leading to an earlier assumption that multiple genes are 32 involved in shaping the overall phenotypic variation at this trait. Through resequencing 33 of 25 representative accessions and allelism tests between light rind accessions, we 34 show that multiple independent SNPs in the CmAPRR2 gene are causative for the light 35 rind phenotype. The multi-haplotypic nature of this gene explain the lack of detection 36 power obtained through GBS-based GWAS and confirm the pivotal role of this gene in 37 shaping fruit color variation in melon. This study demonstrates the power of combining 38 bi-and multi-allelic designs with deep sequencing, to resolve lack of power due to high 39 haplotypic diversity and low allele frequencies. Due to its central role and broad effect 40 on pigment accumulation in fruits, the APRR2 gene is an attractive target for 41 carotenoids bio-fortification of cucurbit crops.42 43