Identification and characterization of candidate genes for peach fruit shape

Abstract

Peach fruit derives from the ripen ovary, developing into drupes whose shape may vary from round to flat, acquiring some intermediates shapes as the oblate. In peach, the flat phenotype is caused by a partially dominant allele in heterozygosis (Ss), fruit from homozygous trees (SS) abort a few weeks after fruit setting. Previous research has identified a SSR marker (UDP98-412) highly associated with the trait, found suitable for marker assisted selection (MAS). Later, an association analysis suggested the putative involvement of a leucine rich receptor-like kinase (LRR-RLK) in fruit shape determination. Specifically, a ~10Kb deletion affecting the promoter and part of the coding region of the gene co-segregated with the flat shape. Here our goal was to clone and study in detail that gene (Prupe.6G281100) to elucidate its function in determining the shape of the fruit. This gene is orthologous to the BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). Protein BLAST alignment identified significant hits with genes involved in different biological processes. Best protein hit occurred with AtRLP12, which may functionally complement CLAVATA2, a key regulator that controls the stem cell population size. RT-PCR analysis revealed the absence of transcription of the partially deleted allele associated with the flat phenotype. The data support Prupe.6G281100 as a candidate gene for flat shape in peach. The posterior screening of larger sample panel has identified three varieties escaping the association between genotype at Prupe.6G281100 and the phenotype. The whole genome sequences of these three varieties revealed additional variability in other two LRR-RLKs genes clustering, together with Prupe.6G281100, in the S locus. Large fraction of this variability accumulated in one of these two genes (Prupe.6G281500 located 21.3 Kb downstream Prupe.6G281100). This variability, in phase with the 10 Kb deletion, was observed in all flat varieties. The second gene with additional variability was Prupe.6G281400 (15.3 Kb downstream Prupe.6G28110) with a 6.1 Kb sequences deleted in phase with the S allele and unique to the outlier varieties. According to gene function prediction, the two LRR-RLKs genes have best homology with FLORAL ORGAN NUMBER1 (FON1) and THICK TASSEL DWARF1 (TD1). These genes have been reported to be involved in mediating floral meristems and organs growth during inflorescence and flower development, including structural organization, shape alteration and size regulation in various species. We formulate here a three allele model hypothesis based on allele conformation at the three genes, which may be involved at different levels of dominance, in fruit shape determination. In this thesis we have also analyzed a somatic mutant with round fruit ‘UFO-4Mut’ derived from the flat variety ‘UFO-4’. Genotypic data revealed that this mutation occurred in cells of the meristematic layer LII. We initiated the study of these two cultivars with the main objective of validating Prupe.6G28000 role in fruit shape determination. However, whole genome analysis revealed a large region (6.5 Mb) in the mutant sample with loss of heterozygosity (LOH). We hypothesize that the LOH may have been produced by the repair of a double strand break (DSB) in the chromosome carrying the allele causing the flat phenotype with the homologous chromosome (carrying the allele for the round). Therefore, the allele producing flat shape has been deleted in the mutant and replaced by the one producing the round shape, which fully explains the phenotype reversion. In this thesis we also initiated protocols for two different techniques. One of them aims at the visualization of genome rearrangements in peach somatic cells by FISH. The other consists on the use of virus vectors to induce gene expression in peach. First steps in the setting of these protocols have been established and are discussed in this thesis manuscript.

Date of Award	28 Nov 2018
Original language	English
Supervisor	Maria José Aranzana (Director)