Transcriptome Sequencing And Molecular Identification Of Genes EjSWEET15 And EjAO From Mutant Loquat

Loquat(Eriobotrya japonica Lindl.)is an economically important crop species originated in China as a characteristic fruit with high medicinal value.Flesh color plays important key roles for loquat phenotype and fruit quality,and Eriobotrya japonica can divided into yellowish-and whitish-fleshed loquat varieties.Whitish-fleshed groups are more favored for their pulps texture tender and sweet taste compared to that yellowish-fleshed loquat.Fruit maturation involves several metabolic changes influencing chemical and textural characteristics,such as color formation and sugar concentration.The variances of flesh color come from differences in carotenoid content of fruit.The mutant loquat described herein resulted in a change of flesh color phenotypes,which is a yellowish-fleshed plant emerged a bud mutation of whitish-fleshed fruit.Now the information of loquat genomic is not clear and the molecular mechanisms of sugar accumulation and fresh-keeping were scarcely reported.RNA-seq is a next-generation sequencing biotechnology that can be used for large scale gene discovery.The objectives of this study were to investigate the variation genes and molecular markers associated with loquat quality by combining RNA-seq with RACE and TR-PCR based on the mutant and the wild loquat.We systematically explored the differentially expressed genes,developed EST-SSR markers and SNPs for the differentially expressed genes,acquiring two genes related to the quality traits of loquat fruits,and made a bioinformatic analysis on these two genes,These study could uncover new insights of be important for flesh quality to understand the gene controlling of sugar biosynthesis,fresh-keeping and other characters in E.japonica,and supply a theoretical platform for studies on quality trait formation,germplasm identification,genetic map construction,gene mapping and cloning,and marker assisted selection in breeding.The concrete study achievements are as follows:1.In this study,we performed RNA-seq of wild red flesh loquat and mutant white flesh loquat samples during four periods(171d,175d,180d and 185d after flower)from fruit coloring to ripening.Roughly 9.5 Gb of Clean reads were obtained with low-quality data removed,and 61,587 Unigenes were assembled(already uploaded to the NCBI Sequence-Read Archive Database,login ID:SUB919374).28,216 differentially expressed Unigenes(13,676 up-regulated ones and 14,540 down-regulated ones)were detected given threshold |log2 Ratio|?1 and probability>0.8.Gene Ontology(GO)and KEGG analyses showed that 40 GO categories and 121 metabolic pathways were significantly enriched,involving secondary metabolite production,plant hormone signaling,oxidative phosphorylation,glycolysis,starch and sucrose metabolism,amino sugar and ribose metabolism,etc.Besides,21 genes related to carotenoid metabolism were identified,and the expression level of most genes was significantly higher in red flesh than in white flesh on the 171d after pollination.2.7,606 EST-SSR sequences were identified by MISA in the 6,608 Unigenes refined from the RNA-seq assembly data of wild red flesh and mutant white flesh loquat fruits.250 pairs of synthetic primers were selected randomly in accordance with annealing temperature,amplified product size and other relevant indexes to amplify the genes of the wild red flesh loquat and mutant white flesh loquat.As a result,clear bands were produced in 226 pairs,the amplification efficiency reaching up to 90.4%.This shows that EST-SSR markers could be developed easily on the RNA-seq data platform of loquat fruits.3.The full-length RNA-seq assembly sequences of loquat fruits were analyzed by GATK,with 2,396 SNPs identified in 1,361 Unigenes,suggesting that the main mutation type was AT,which mutated into GC(768),followed by GC,which mutated into AT(726)as base transition.The frequency of base transition was 1.65 times as high as that of base transversion.The frequency of occurrence of SNP in Unigenes was 2.2%,and most(roughly 87%)SNPs were nonsense mutations,while 311 were missense mutations.It was discovered that 11 SNPs in the 3 candidate genes were related to carotenoid biosynthesis in the loquats.at least one of which was a missense mutation that led to a change in coded amino acids.4.SWEET protein is a kind of sugar transporter discovered in eucaryotes in recent years,which has two typical MtN3/saliva domains.By combining rapid amplification of cDNA end(RACE)with RT-PCR,we successfully cloned a SWEET protein-coding gene in the mature fruits of white flesh loquat,named EjSWEET15.The full length of this gene was 1,271 bp,containing an open reading frame with a length of 918bp.This open reading frame encoded 305 amino acids,and formed 34.13 kDa of predicted protein molecular weights.The result of real-time fluorescence quantification PCR showed that EjSWEET15 was highly expressed in leaves and fruits,and its expression level was much higher in white flesh loquat fruits than in red flesh loquat at the maturity stage.Nucleotide diversity analysis showed that there were 4 polymorphic sites for EjSWEET15 in red flesh loquat and white flesh loquat,1 of which was in the promoter region,while the rest 3 were in the open reading frame,all of which led to changes in amino acids,and this might exert an effect on the enzyme activity of EjSWEET15.The cloning of EjSWEET15 and the identification of polymorphic sites might serve as a theoretical foundation for illustrating the formation mechanism of loquat fruit quality traits as well as its genetic improvement and breeding.5.Fruit maturation and senescence form a complicated biological process,in which reactive oxygen species(ROS)is generally recognized to play a key role.In this study,by combining RACE with RT-PCR,we cloned an ascorbic acid oxidase(AO)-encoding gene in the mature fruits of white flesh loquat,named EjAO.The full length of this gene was 2,174bp,containing an open reading frame with a length of 1,605bp.This open reading frame encoded 534 amino acids,and formed 59.39 kDa of predicted protein molecular weights.The result of real-time fluorescence quantification PCR showed that EjAO was highly expressed in leaves and fruits.and its expression level was much higher in white flesh loquat fruits than in red flesh loquat at the maturity stage.Nucleotide diversity analysis showed that there were 5 polymorphic sites for EjAO in red flesh loquat and white flesh loquat,among which both T719C and A730C led to changes in amino acids,and this might exert an effect on the enzyme activity of EjAO.The cloning of EjAO and the identification of polymorphic sites might serve as a theoretical foundation for illustrating the maturation and senescence mechanism of loquat fruits as well as the genetic improvement and breeding of loquat.