Differentially Expressed Gene Analysis Of Apple Anther Under Low Temperature Induction

Anther culture is one of the main methods of apple haploid breeding,which is the process of forming the whole plant by means of embryoid or an organ.Through embryoid culture,it can not only directly differentiate into plantlets,but also has a short time of seedling,high survival rate,and high regeneration efficiency.A certain period of low temperature treatment before apple anther culture can induce the anther to directly form the embryoid and not through callus.In this study,the research on the apple anther that were treated by low temperature and the embryoid and the callus that were formed after low temperature treatment,was carried out by the method of RNA-Seq.The differentially expressed genes in the process of embryoid formation were analyzed and genes related to the embryogenic potential of apple anther after low temperature induction were screened,and the relationship between the related genes and the formation of the embryoid was discussed.The results of the study are as follows:1.The anther of apple cultivar 'Gala' was used as material to study the effect of different low temperature treatment time on the apple anther from the gene level.6 samples were determined:T1(untreated anther),T2(anther after 10 days low temperature),T3(anther after 30 days low temperature),T4(anther after 30 days low temperature and in culture medium for 30 days),T5(embryoid formed by anther that was treated by 30 days low temperature),T6(callus formed by anther that was treated by 30 days low temperature).2.34.88 Gb clean data was obtained from 6 samples by sequencing,The clean data of each sample reached 5.4 Gb,percentage of Q30 bases in 89.08%and above.Clean Data of each sample was blasted to the reference genome sequence,and the efficiency percentage was 74.4%and above.The sequencing quality of the 6 samples were all satisfied with the experimental requirements.3.According to the experimental results,the comparison group was determined as follows:T1-T2,T1-T3,T1-T4,T5-T6.Through gene expression analysis,21,851 differentially expressed genes were obtained,including 10,302 up-regulated genes and 11,549 down regulated genes.4.The GO classification showed that the number of differentially expressed genes in the process of 'biological process' was the most,and the highest proportion of differentially expressed genes was expressed in the'cellular process' and 'metabolic process'.COG orthologous classification results showed that differentially expressed genes in R(general function prediction)are the most common,followed by K(transcription),L(replication,recombination and repair),T(signal transduction mechanism),G(carbohydrate transport and metabolism).KEGG classification showed that the metabolic pathways of differentially expressed genes were most concentrated in the 'metabolism'.The significant enrichment of differentially expressed genes was mainly related to hormone signal transduction and carbohydrate metabolism,and the most enriched metabolic pathway was'plant hormone signal transduction' and 'starch and sucrose metabolism'.5.In the group of T1-T2,T1-T3,T1-T4,the expression of DEG which control sucrose biosynthesis,cytokinin,abscisic acid,gibberellin and brassinosteroid's signal transduction were up-regulated while those control starch biosynthesis and auxin's signal transduction were down-regulated.In the group of T1-T4,T6-T5,the expression of DEG which control sucrose and starch biosynthesis all showed a decrease after the first rise,and DEG which control auxin's signal transduction were down-regulated while those control cytokinin,gibberellin and abscisic acid's signal transduction were up-regulated.6.The RT-qPCR results showed the sequencing results were consistent with the actual results.Therefore,the differentially expressed genes screened in this study improved embryoid formation rate through the increase in expression of genes related to sucrose,cytokinins,abscisic acid,gibberellin,brassinolide and the decrease in expression of genes related to starch and auxin.As a result,change of the expression of the genes screened in this study is the key to the embryogenic potential of apple anther after low temperature induction.