‘艳丽’草莓微繁殖苗与普通苗的表型差异及转录组分析

Phenotypic variation between micro-propagated and conventional plants of‘Yanli’strawberry and its mechanism revealed by transcriptome analysis

【目的】分析草莓微繁殖苗与普通苗表型差异,通过转录组测序揭示草莓微繁殖苗与普通苗差异表达的基因,探索草莓微繁殖苗与普通苗表型差异的调控机制。【方法】以草莓品种‘艳丽’为试材,在繁苗期和栽培期调查微繁殖苗与普通苗之间的表型差异,并以茎尖生长点为试材进行转录组测序分析。【结果】在繁苗期,微繁殖原种苗的匍匐茎数、新茎数、叶片数均明显高于普通苗,而株高、冠径、叶面积差异不明显。在栽培期,微繁殖一代苗比普通苗开花结果略早,但是两者在果实大小、产量、可溶性固形物含量上没有明显差异。微繁殖原种苗与普通苗的茎尖生长点里有1803个基因显著差异表达,相对于普通苗,微繁殖原种苗中上调表达基因1469个,下调表达基因334个。差异表达的基因涉及光合作用、植物激素合成及信号转导、植物病原菌互作等。【结论】‘艳丽’草莓微繁殖苗的繁苗能力明显高于普通苗,这一表型差异与植物激素合成及信号转导通路上基因的差异表达有关。

【Objective】Strawberry,the perennial herb of the family Rosaceae,is widely cultivated in the world due to high economic and nutritional value.Currently,the area of cultivation of strawberry is increasing rapidly in China.Strawberry plants are mainly propagated by runners which would lead to spreading of some diseases.Therefore,micro-propagation technology has been used to generate diseasefree plants to improve the quality of products.Some studies have reported that micro-propagated strawberry plants have more runners,leaves,and even high net photosynthetic rate.However,the molecular mechanism of the differences is rarely reported.In this study,we investigated the differentially expressed genes between the micro-propagated and the conventional plants of strawberry cultivar‘Yanli’through transcriptomics analysis.【Methods】The micro-propagated stock plants of cultivated strawberry(Fragaria×ananassa Duch.)cultivar‘Yanli’were transplanted and were compared with the conventional plants propagated by runner mode in the field.Some of the investigations and analysis were as follows:(1)Investigation of morphological indexes during the vegetative propagation stage.The micropropagated stock plants and the conventional plants were planted in the pots in open field,and the number of leaves,branch crowns,runners,etc.were recorded every 14 days.(2)Phenological observation.The first generation of the micro-propagated stock plants and the conventional plants were cultivated in solar greenhouse,and the growth and development status were investigated every 3 days and the first flowering date,flower blooming date,and the fruit ripening date were recorded.(3)Investigation of morphological indexes during the flowering and fruiting stage.Plant height,plant diameter,number of leaves and leaf area were measured from 30 plants in the period of the beginning of the flowering,fruit ripening,and the end of the first fruit harvest.(4)Fruit yield and quality.The hardness and soluble solid content of the first fruit were measured through a hardness tester and a refractometer,besides,the weight of single fruit was measured through an electronic scale.(5)Transcriptomics analysis.The shoot tips of the micro-propagated‘Yanli’and the conventional plants cultivated in the open field for 30 days,were used for the RNA-seq.The library was constructed and clean reads were obtained by removing reads with 3’-end adapters,poly-N(N indicates that the base information cannot be determined)(≥10%),and low-quality sequences(Q≤10 bases account for more than 50% of the total sequence bases).A short sequence assembly software Trinity was used to perform de novo assembly and the longest transcript as unigene was selected for subsequent annotation,quantification,and differential expression analysis.Here,differential expression genes(DEGs)were screened by threshold|log2Fold Change|>1 and corrected p-value<0.05.To explore the gene function,multiple protein databases(BLAST+/Uniprot),protein domain Recognition(HMMER/PFAM),pathway function(GO/KEGG databases),homologous protein clustering(eggNOG)were used to annotate unigene CDS,and NR database,Rfam database,plant-related Arabidopsis database were used to annotate unigene sequences of unpredicted CDS.【Results】Among morphological indicator difference between the micro-propagated and the conventional plants,the number of runners of the micro-propagated plants increased by 3-4 times,leaves increased by 1.8 times but new stems decreased during the vegetative propagation stage compared with those of the conventional plants.By transcriptome sequencing,a total of 236717178 clean reads were obtained then the 55.6% predicted unigenes and 63.2% unpredicted unigenes were annotated according to the above databases.Among the 1803 DEGs,1469 DEGs were up-regulated,while 334 were down-regulated in the shoot apical meristems of the micro-propagated plants.According to GO analysis,the DGEs were annotated by molecular function,biological process and cellular component.The top 20 groups with the most differences were selected to analyze.Here,sucrose synthase,sucrose metabolism pathway,hormone metabolism,alcohol dehydrogenation enzymes,etc.were the most significantly downregulated,while chitinase,organic nitrogen compounds,jasmonic acid,etc.were the most significantly up-regulated.According to KEGG analysis,the top 15 paths with a large number of annotation genes were classified into four groups:metabolism,genetic information processing,cellular processes,and environmental information processing.The most significant pathways were photosynthesis-antenna,plant hormone signal transduction,isoquinoline alkaloid biosynthesis,terpenoid backbone biosynthesis,linoleic acid metabolism.Among the 27 DEGs of plant hormone signal transduction,5 DEGs of auxin-responsive protein,four TIFYs,and the two jasmonate related genes JAZ10 and JAZ1 were up-regulated in the micro-propagated plants.The expressions of arginine biosynthesis,zeatin biosynthesis,tyrosine metabolism genes were all down-regulated in the micro-propagated plants.Also,some specific genes were analyzed,such as the GA20x,the WRKY33 which were up-regulated.Strigolactone synthesis gene CCD7 was also up-regulated in the micro-propagated plants.Those differential expression genes might cause the difference between the micro-propagated plants and the conventional plants.【Conclusion】In this study,the number of runners and leaves of the micro-propagated plants were significantly higher than those of the conventional plants,and the number of branch crowns was significantly lower.Transcriptome sequencing analysis showed that the genes related to plant hormones were significantly and differentially expressed.It showed that hormone signaling played a key role in the morphological development of the micro-propagated plants.