A CACTA transposable element in a PpYUC11 gene promoter is associated with the stony hard phenotype in peach

Abstract：【Objective】The fruits of melting-flesh peach produce high levels of ethylene induced by highconcentration of IAA, resulting in rapid fruit softening at the late-ripening stage. In contrast, the fruits ofstony hard peach produce few ethylene due to the inhibition IAA Sythesis. YUCCA encodes flavin monooxygenase-like enzyme, and converts indole-3-pyruvic acid to IAA. In our previous study, a YUCCA fla⁃vin mono-oxygenase gene (PpYUC11, ppa008176m) displayed an identical differential expression profileto the profiles of IAA accumulation during peach fruit ripening. The strong association between intron TCmicrosatellite genotypes of PpYUC11 and the flesh texture (normal or SH) was described in 43 peach vari⁃eties, indicating that this locus might be responsible for the SH phenotype in peach. In this study, the rea⁃son leading to the different expression level of PpYUC11 in melting-flesh and stony hard peach duringfruit ripening was explored.【Methods】Quantitative PCR analysis was performed on the expression levelsof PpYUC11 in the fruits and seeds during fruit ripening, and the upstream region of PpYUC11 gene of SHand non-SH peach cultivars was isolated. The upstream regulatory cis elements responding to plant hormone were investigated using PlantCARE, and cis element of the fruit ripening-related in the PpYUC11promoter sequence was manually searched.【Results】PpYUC11 showed increasing expression in the MF‘( Zhongyoutao 13’) fruits during fruit ripening, and peaked at S4 III, but the expression was very low inthe SH‘( Zhongyoutao 16’) fruits. Unlike in the fruits, the expression of PpYUC11 was high both in theMF‘( Zhongyoutao 13’) and the SH‘( Zhongyoutao 16’) seeds. To explore the reason leading to the difference of the expression in the MF and the SH fruits, we cloned the upstream predicted promoter region ofthe PpYUC11 gene in the MF‘( Zhongyoutao 13’and‘Gold honey 3’) and the SH‘( Zhongyoutao 16’and‘Yumyeong’). A large insertion was found in the 2.1 kb upstream from the start codon of thePpYUC11 gene in the SH peaches. The insertion fragment was 2 572 bp long, and highly homologous topeach PpDAM6 gene which was published on the NCBI database. This fragment was a typical CACTAtype DNA transposon, with terminal inverted repeats (CAAGAAAAAA) and conserved sentence (CAC⁃TA), and contained 2 539 bp transposon sequence. There were 8 loci with high homology with this CAC⁃TA transposon in the peach genome, being located on 1 (1), 2 (2), 3 (1), 4 (3), 7 (1) chromosomes, thescore (Blast score) was more than 3 000.0 and the E value was zero. To investigate the relationship between the CACTA transposon of PpYUC11 and the phenotype of flesh texture (normal or stony hard), twoprimers were designed to detect the insertion of CACTA transposon at the PpYUC11 locus in 26 normaland 6 SH flesh cultivars. The CACTA transposon appeared to be associated with SH cultivars when it pre⁃sented in a homozygous state, whereas normal-fleshed (M or NM) cultivars possessed at least another al⁃lele at the PpYUC11 locus. These cultivars belonged to three genotypes: the genotype of‘Hakuto’,‘Okitsu’‘Hangongzhu’‘Okubao’‘Goldhoney 3’were Hd/hd; the genotype of all the stony hard flesh culti⁃vars‘( Zhongyoutao 16’‘Xiacui’‘Huayu’‘Jingyu’‘Qingwang’and‘Yumyeong’) were hd/hd, and thegenotype of other cultivars were Hd/Hd. Seven types of cis elements responding to plant hormones werepresented in the 2.5 kb upstream sequences of the PpYUC11 gene promoter region, and there was a fruitspecificelement around 100 bp upstream of the insertion site. It was suggested that the fruit-specific element would be involved in the tissue specific expression of PpYUC11, whereas the insertion of the CAC⁃TA transposon might disturb the element binding to the transcription factor that was associated with fruitmaturation, resulting in the low expression level of PpYUC11 in the SH peaches fruits during fruit ripen⁃ing.【Conclusion】The expression of PpYUC11 was inhibited in the SH peach fruits, and was not inhibitedin the SH peach seeds. This specific expression might be due to the insertion of the CACTA transposon,however, further promoter deletion analysis is needed to qualify the mutation mechanism associated withthe stony hard phenotype. Stony hard peaches are characterized by the absence of both ethylene produc⁃tion and softening in mature fruits and are expected to be used as a genetic source for breeding new tablepeaches. However, stony hard fruits are often very difficult to distinguish from the NM or very firm, unripe M phenotypes in the field, resulting in the difficulty in selection. Genotyping the CACTA transposonof PpYUC11 could be used for the marker-assisted breeding of new cultivars of the stony hard fleshpeach.