Genetic characterization of the Japanese plum LG3-MYB10 region and development of molecular markers for fruit color

Abstract

Japanese plum (P. salicina and its hybrids) is a highly heterozygous fruit tree originated by the interspecific hybridization of more than eleven diploid plum species. This event might explain that, within the Rosaceae family, it is one of the crops with the widest fruit color variability. Despite the economic importance of Japanese plum fruits in the fresh market, its marker-assisted breeding is still in its infancy. This applies for the fruit color trait, which influences the consumer acceptance and, consequently, is included within the key breeding objectives. The red to black hues are caused by the accumulation of anthocyanins, pigments synthesized from the flavonoid pathway that have nutraceutical properties. The MYB10 genes are candidates for anthocyanin accumulation in plant organs, and QTLs for red fruit color in Prunus have been mapped in linkage group 3 (LG3) in a region containing three gene copies in peach: PpMYB10.1, PpMYB10.2 and PpMYB10.3. The objective of this thesis is to study the role of the LG3-MYB10 genes in the regulation of fruit color in Japanese plum and provide efficient molecular markers for marker-assisted breeding. To study the allelic variability of the Japanese plum LG3-MYB10 genes (PsMYB10), we used a set of primers designed in conserved sites of the peach PpMYB10 genes by using its genome assembly (Chapter 1). We found one allele (a356) highly associated with the fruit skin color, but none for the flesh. By means of progeny segregation and allele cloning we assigned the alleles into haplotypes and identified a PsMYB10.1 triplication (Chapter 2). Allele a356 was a PpMYB10.1 homolog, with allele a470 segregating as an alternative allele, recessive for the locus (PsMYB10.1a). To sequence the LG3-PsMYB10 region, we used a novel long-read targeted sequencing technology, the CRISPR-Cas9 enrichment followed by Oxford Nanopore Technology sequencing (Chapter 3). Despite the target region was large, highly variable and with several duplicated genes, with our strategy we could extract polymorphisms from the genome alignments against reference genomes and from de novo aligned sequences. All these previous results served to find association of the LG3-MYB10 region with the red flesh color (Chapter 4), identifying a retrotransposon in the PsMYB10.2 gene correlating with its expression. The gene function was validated by setting up a protocol for its transient overexpression in Japanese plum fruits. The main outputs of this thesis are 1) the development of efficient molecular markers for the fruit skin and flesh colors, 2) the validation of a strategy for the CRISPR-Cas9 enrichment of large and highly variable regions with gene duplications in a pool of samples, applicable in other plant and animal studies, and 3) a protocol for the transient gene overexpression in Japanese plum fruits, applicable for the validation of other candidate genes in the crop.

Date of Award	5 May 2022
Original language	English
Supervisor	Aranzana Civit, Maria Jose (Director)