Identification And Functional Analysis Of Key Genes For Fruit Cracking Based On Whole Genome And Transcriptome In Chinese Jujube

Chinese jujube is a unique economic tree species that originates from China.Fruit cracking severely affects the production and quality of jujube and restricts the development of jujube industry.At present,a lot of studies have been carried out on the physiological and biochemical characteristics of jujube fruit cracking,and it is recognized that fruit cracking is a genetic trait controlled by genes,but little is known about the cracking-related molecular mechanisms.It is very important for improving jujube cultivars to further identify genes related to fruit cracking through multi-omics analysis.Based on the comprehensive evaluation of different applied methods of fruit cracking,the high-quality single nucleotide polymorphisms（SNPs）of jujube germplasm collections were used to conduct genome-wide association analysis（GWAS）with fruit cracking traits.The extremely cracking-susceptible/resistant cultivars and F₁individuals were used as experimental materials.According to the variation of plant hormone metabolites,the differentially expressed genes（DEGs）in the key pathways of jujube fruit cracking were analyzed from the transcriptome.Bioinformatics analysis of cell wall related genes were carried out,and the functions of genes were studied in tomato.The main results and conclusions are as follows:（1）The results showed that the methods of indoor immersion and field test were more suitable for the determination of fruit cracking traits of jujube germplasm collections.By comparing the different applied methods of fruit cracking,the current data revealed that waterfall method exhibited lower distinctive ability for cracking evaluation.The cracking resistance of jujube cultivars can be accurately determined by vacuum-immersion under the pressure of 33.33KPa,decompression for 5min and immersion for 4h.There were no significant differences in cracking rate and cracking index between the methods of vacuum-immersion and indoor immersion for 24h.（2）GWAS was used to detect SNPs significantly associated with fruit cracking traits using high-quality SNP markers developed by genotyping-by-sequencing（GBS）and resequencing.A total of 4651high-quality SNP markers were identified in 180 jujube accessions by GBS sequencing.The GWAS conducted using the MLM model identified three significant SNPs and two candidate genes.The fruit cracking traits of 300 jujube accessions were measured.Correlation analysis showed that there were significant correlations between fruit cracking traits and texture traits.A total of 213 significant SNPs,8significant SNP signal clusters and 93 candidate genes were identified by GWAS using resequencing-derived SNP markers.（3）The DEGs involved in the pathway of fruit cracking were obtained by transcriptome analyses in jujube cultivars and F₁ individuals with differential resistance to fruit cracking.The comparative transcriptome analyses of cracking and normal fruits of cracking-susceptible and-resistant jujube cultivars.A total of 131 DEGs were common to the cracking‘Cuizaohong’vs.normal‘Cuizaohong’and cracking‘Jinsixiaozao’vs.normal‘Jinsixiaozao’comparisons.Trend analysis showed that the expression levels of 225 DEGs were continuously downregulated in the cracking‘Jinsixiaozao’,normal‘Jinsixiaozao’,and normal‘Muzao’samples.A total of 61 cracking-related genes were identified by functional annotation.Transcriptome analyses of exocarp and mesocarp in four developmental stages of cracking-susceptible and-resistant F₁individuals were conducted.There were 121 and 128 cracking-related genes in exocarp and mesocarp,respectively.（4）Combined GWAS and transcriptome analysis,a total of 14 cracking-related genes were obtained by association analysis.Transcription factors WRKY23 was highly expressed in the mesocarp of cracking-resistant individual,and the other 13 genes were highly expressed in the cracking-susceptible samples or cracking fruits.Expression analysis showed that four genes Zj GATL5,Zj LACS1,Zj NPF4.2 and Zj OPT3were highly expressed in fruit.（5）ABA,JA and ACC play an important role in regulating fruit cracking,among which ABA and JA mainly regulate the cell wall pathway and promote fruit cracking.A total of 14 plant hormone metabolites in exocarp and mesocarp of cracking-susceptible and-resistant individuals were detected by HPLC-MS/MS.During the fruit development,the ABA and JA contents in exocarp of cracking-susceptible individual were significantly higher than those of cracking-resistant individual,and the ACC contents in exocarp and mesocarp of cracking-resistant individual were significantly higher than those of cracking-susceptible individual.Applications of exogenous ABA and Me JA significantly increased fruit cracking.RNA-seq analysis showed that more DEGs were enriched in GO items of cell wall.Among the transcription factors,the most abundant DEGs were AP2/ERF-ERF family members.（6）A total of 30 Zj EXP genes were identified in the‘Dongzao’,and 12 Zj PG genes were expressed in the exocarp of cracking-susceptible and-resistant individuals.Using transcriptome data and q RT-PCR,Zj EXPA6 and Zj PG5 were highly expressed in fruit,and differentially expressed in the exocarp of cracking-susceptible and-resistant individuals during the high incidence cracking stage.（7）The overexpression of cell wall related genes Zj GATL5,Zj EXPA6 and Zj PG5 in tomato can significantly enhance the activity of cell wall metabolic enzymes in fruit,and then resist or promote fruit cracking.Overexpression of Zj GATL5 significantly enhanced the activities of polygalacturonase and cellulase,and increased the total soluble solids in fruit.Overexpression of Zj EXPA6 and Zj PG5 genes significantly enhanced the activity of polygalacturonase in fruit.The fruit color of tomato overexpressed with Zj PG5 became lighter.In this study,the molecular mechanism of jujube fruit cracking was preliminarily revealed,the key genes related to fruit cracking were identified,and the effects of Zj GATL5,Zj EXPA6 and Zj PG5 on the fruit cracking-related traits were confirmed using jujube accessions and F₁ individuals through the multi-omics and molecular biology methods.The results lay a foundation for molecular maker assisted breeding of cracking resistance in jujube.