Identification Of The Mutation Locus And The Molecular Mechanism Of The Pear Red Sport Of ’Zaosu’

Red-skinned pear is deeply favorite by consumers for its attractive appearance and nutritional value,but the red-skinned pear cultivars are few in China.In production,many red-skinned pear cultivars were selected from the sports of green cultivars,but the mutation loci and their coloring mechanisms are unclear yet.Studying the coloring mechanism of red trait will be very important in the breeding and growing regulation of red-skinned pear.In this study,a red sport cultivar ‘Zaosu Red’,which was selected from an early-maturing,crisp and domestically bred cultivar ‘Zaosu’,was used as the main material to study its coloring mechanism,the results are as follows:1.The anthocyanins in young leaves and fruit peels of ‘Zaosu Red’ were mainly composed of cyanidin-3-O-galactoside,cyanidin-3-O-glucoside,cyanidin-3-O-arabinoside and peonidin-3-O-galactoside,the proportion of different types of anthocyanins between leaves and fruit peels were slightly different,the biosynthesis pathway of anthocyanins between leaves and fruits may be different;light is necessary for fruit coloring of ‘Zaosu Red’,visible light+UV-B treatment could make the bagged fruits of ‘Zaosu’ turn red;the genes Pp ANS,Pp MYB10 and Pp HY5,which are important in anthocyanin biosynthesis and regulation,had a relatively higher expression in the red samples than that in green samples,and were the key genes in coloring of ‘Zaosu Red’;the promoter sequences of these genes were consensus between‘Zaosu’ and ‘Zaosu Red’,and their expression differences were not caused by the sequence variant.2.A 510.59 Mb high quality pear genome was assembled by combining the next-generation,third-generation and Hi-C sequencing technologies using the pear dwarfing rootstock cultivar ‘Zhongai 1’,with a contig N50 size of 1.28 Mb,which covered 99.86% of the estimated genome;506.31 Mb(99.16%)of the contigs were clustered into 17 chromosomes,of which 427.8 Mb were located and oriented,covered83.7% of the estimated genome;we further predicted 309.86 Mb(60.68%)of repetitive sequences and43,120 protein-coding genes;the results of sequences alignment,core genes test and collinearity analysis showed that the integrity and accuracy of our assembled genome is well.3.Using the newly assembled pear genome,by combining bulked segregant analysis with whole-genome sequencing,we identified a 14 nucleotide deletion mutation from the red pear mutant‘Zaosu Red’,which was closely associated with the red trait of ‘Zaosu Red’.This 14 bp deletion was only identified in ‘Zaosu Red’ and its red progeny,and didn`t appeared in other test 16 red and green cultivars/clones,which implied that differentially sourced red-colored pears have various types of coloring mechanism.4.The Pp BBX24 gene was cloned from ‘Zaou’ and ‘Zaosu Red’,and the result of sequencing showed that a 14 bp deletion is in Pp BBX24 gene,which was consistent with the result of genome sequencing and BSA.The full coding sequence of Pp BBX24 gene is 720 bp and encoded 239 aa.The 14 bp deletion variant identified from ‘Zaosu Red’ is located at 568 bp to 581 bp of the coding region of Pp BBX24 gene,whichcauses a coding frame shift and early termination,and only 205 aa are translated.At the C termination of the early terminated Ppbbx24-del,an important nuclear localization sequence and a VP domain which is necessary for protein-protein interaction are lost,which caused the Ppbbx24-del losing the interaction activity with Pp HY5 a,Pp HY5 b and Pp PUB59.The transcript levels of Pp BBX24 gene between ‘Zaosu Red’ and ‘Zaosu’ were not significantly different,because the Pp BBX24 gene has two types of normal and mutant in ‘Zaosu Red’,the transcript amount of the normal Pp BBX24 gene in ‘Zaosu Red’ was only a half of that in ‘Zaosu’,which may be the key reason for the red coloring of ‘Zaosu Red’.