Study On The Morphological Characteristics And The Forming Mechanism Of 'Liehe' Apricot Endocarp

Apricot(Prunus.armeniaca L.)is a typical drupe of the family Rosaceae with eight chromosomes(2 n = 16).A complete and hardened endocarp is a typical trait of drupe fruits.China has a great wealth of germplasm genetic resources of apricot that have important breeding values.'Liehe'(LE)apricot is an extremely rare cultivar that originated in Linyuan City of Liaoning Province.'Liehe'(LE)apricot cultivar has a thin,soft,cleavable endocarp that represents 60.39%of the thickness of the 'Jinxihong'(JG)apricot(with normal hardened-endocarp).Current status of the endocarp study already have some basis,however LE apricot just exist in China.Thus,it's first time to research the endocarp development of LE apricot.In this study,LE apricot was mainly experimental material,and JG apricot was always control sample.We have determined the phenological period of apricot fruit development,and observed the endocarp lignification.Then,we investigated the mechanism of endocarp development in LE apricot through mining differentially expressed genes from RNA-seq,analysising the gene expression level during fruit development,cloning CAD and NST1 genes,testing genes expression in different kernel-using apricot.The main results were as follows:1.Based on Pearl-Reed logistic model,the growth curves of LE and JG apricot were calculated.Combining with expression levels of ACO1 and PEPCK,the phenological period of two apricots fruit development were divided into same four stages(S1-S4).The processes of endocarp development of two apricots were almost the same.It was close to 25 d for lignin deposition.At 15 days after full bloom(DAFB),endocarp of LE apricot begun to cleave.There were significant correlation between endocarp thickness and lignin content.The thickness of LE endocarp was thinner than JG endocarp.For endocarp textures,such as crispness,firmness,cohesiveness and adhesiveness were 113%,46%,73%and 527%in LE apricot compared with JG cultivar at 42 DAFB,respectively.2.Comparative transcriptomes of Prunus armeniaca L.were sequenced using Illumina HiSeqTM 2500 to reveal the molecular mechanisms behind the LE apricot phenotype,The LE endocarp cleaving occurred at 15 DAFB and rapidly increased at 21 DAFB.Thus,RNA from these two fruits stages of LE and JG apricot was used for RNA-seq,with two replicates per fruit,which generated 40,145,230,606 raw reads.Transcripts assembled 63,170 genes from LE and JG apricot.Based on the sequence similarity,25,356 genes were matched to NR databases.KEGG enrichment analyses of DEGs showed that Phenylalanine pathway had obvious influence,and 34 DEGs involved in Phenylalanine pathway.CAD gene expression levels,encoding cinnamyl alcohol dehydrogenase,were only 1.3%,0.7%,0.2%and 2.7%in LE apricot compared with JG cultivar at 15,21 DAFB.CAD expression and endocarp thickness had significant correlation at 5%levels.3.CAD and NST1 coding sequences(CDS)of LE and JG apricot were obtained by homologous clone from RNA-seq data,and CDS length were 1092 bp and 1187 bp.For NST1,encoding 395(aa),had 2 difference amino acid.Differential amino acid sites were located at motif 3 and motif 6.For CAD,encoding 363 amino acid(aa),had 5 difference amino acid.Secondary and tertiary structure prediction of CAD encoding amino acid sequence represented that the structures of differential amino acid as 122 aa,127 aa,354 aa were coils;the structures of differential amino acid as 294 aa 343 aa were a-helixes.4.During S2 stage,cinnamyl alcohol dehydrogenase activity of JG apricot was continued to rise.However for LE apricot,it was rised firstly,and then decreased.The endocarp thickness had 5%level coorelation with CAD expression in two apricots,while it also had 1%level coorelation with enzymes activities of CAD in LE apricot.The CAD expression level of fruit of JG apricot was higher than LE apricot,the similar regulation to NST1 expression level except 33 DAFB.5.Enzymes activities of LAC,POX,PAL in LE and JG apricot were detected.Meanwhile,the expression levels of LAC4,PER72,PAL were analysised.During S1 stage,enzymes activities of LAC,POX and expression levels of LAC4,PER72 were much higher in JG apricot than LE apricot,while PAL expression of LE apricot was up-regulated compaired with JG apricot.Enzymes activities of LAC,POX had significant correlation with endocarp lignin content respectively at 5%level in LE apricot.In JG apricot,LAC activity and endocarp lignin content had significant correlation at 5%level.6.The differences of endocarp thickness of kerner-using apricot in this study were significant.We had used 10 factors related to endocarp to applied principal component analysis(PCA)with the SPSS(19.0)software.Contribution rate of PCA 1 was the highest(38.69%),and the cumulative contribution rate of the first PCA 1-5 was up to 94.78%,representing almost factors.Comprehensive rank of endocarp among severial kernel-using apricots(high to the end)were Longwangmao(LWM),JG,seedling progeny of LE,Baiyubian(BYB),Yiwofeng(YWF),Weixuan No.1(W1),LE and You 1(Y1).Beside,the rank of kernel-using apricot based on thickness of endocarp(from thick to thin)were BYB,JG,LWM,YWF,seedling progeny of LE,LE,W1,LE and Yl.These two ranks were almost the same,and PCA analysis could distinguish the apricot which endocarp was thick or thin.LE apricot had crisper,less lignified endocarp that adhered tightly with flesh.Based on these results,the main reasons for endocarp differences between LE and JG apricot were testure and lignin deposition.CAD and NST1 may have colse relations with these differences.