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

ZENG Wenfang; DING Yifeng; PAN Lei; WANG Xiaobei; NIU Liang; LU Zhenhua; CUI Guochao; WANG Zhiqiang

doi:10.13925/j.cnki.gsxb.20170060

Abstract：[Objective] The fruits of melting-flesh peach produce high levels of ethylene induced by high concentration of IAA,resulting in rapid fruit softening at the late-ripening stage.In contrast,the fruits of stony 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 flavin mono-oxygenase gene (PpYUCll,ppa008176m) displayed an identical differential expression profile to the profiles of IAA accumulation during peach fruit ripening.The strong association between intron TC microsatellite genotypes of PpYUC11 and the flesh texture (normal or SH) was described in 43 peach varieties,indicating that this locus might be responsible for the SH phenotype in peach.In this study,the reason leading to the different expression level of PpYUC11 in melting-flesh and stony hard peach during fruit ripening was explored.[Methods] Quantitative PCR analysis was performed on the expression levels of PpYUC11 in the fruits and seeds during fruit ripening,and the upstream region of PpYUC11 gene of SH and 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 PpYUC11 promoter 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 in the SH (‘Zhongyoutao 16’) fruits.Unlike in the fruits,the expression of PpYUC11 was high both in the MF (‘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 of the 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 the PpYUC11 gene in the SH peaches.The insertion fragment was 2 572 bp long,and highly homologous to peach PpDAM6 gene which was published on the NCBI database.This fragment was a typical CACTA type DNA transposon,with terminal inverted repeats (CAAGAAAAAA) and conserved sentence (CACTA),and contained 2 539 bp transposon sequence.There were 8 loci with high homology with this CACTA transposon in the peach genome,being located on 1 (1),2 (2),3 (1),4 (3),7 (1) chromosomes,the score (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),two primers were designed to detect the insertion of CACTA transposon at the PpYUC11 locus in 26 normal and 6 SH flesh cultivars.The CACTA transposon appeared to be associated with SH cultivars when it presented in a homozygous state,whereas normal-fleshed (M or NM) cultivars possessed at least another allele 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 cultivars (‘ Zhongyoutao 16’ ‘ Xiacui’ ‘ Huayu’ ‘ Jingyu’ ‘ Qingwang’ and ‘ Yumyeong’) were hd/hd,and the genotype of other cultivars were Hd/Hd.Seven types of cis elements responding to plant hormones were presented in the 2.5 kb upstream sequences of the PpYUC11 gene promoter region,and there was a fruitspecific element 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 CACTA transposon might disturb the element binding to the transcription factor that was associated with fruit maturation,resulting in the low expression level of PpYUC11 in the SH peaches fruits during fruit ripening.[Conclusion] The expression of PpYUC11 was inhibited in the SH peach fruits,and was not inhibited in 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 with the stony hard phenotype.Stony hard peaches are characterized by the absence of both ethylene production and softening in mature fruits and are expected to be used as a genetic source for breeding new table peaches.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 transposon of PpYUC11 could be used for the marker-assisted breeding of new cultivars of the stony hard flesh peach.