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

Abstract:【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 in- creasing 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 disease- free plants to improve the quality of products. Some studies have reported that micro-propagated straw- berry 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 ex- pressed 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 conven- tional 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 micro- propagated stock plants and the conventional plants were planted in the pots in open field, and the num- ber 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 remov- ing 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 tran- script 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+/Uni- prot, protein domain Recognition (HMMER/PFAM), pathway function (GO/KEGG databases), homolo- gous protein clustering (eggNOG) were used to annotate unigene CDS, and NR database, Rfam data- base, plant-related Arabidopsis database were used to annotate unigene sequences of unpredicted CDS.【Results】Among morphological indicator difference between the micro-propagated and the convention- al 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 236 717 178 clean reads were obtained then the 55.6% predicted unigenes and 63.2% unpredicted unigenes were annotated according to the above databases. Among the 1 803 DEGs, 1 469 DEGs were up-regulated, while 334 were down-regu- lated 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 path- way, hormone metabolism, alcohol dehydrogenation enzymes, etc. were the most significantly down- regulated, 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 en- vironmental information processing. The most significant pathways were photosynthesis-antenna, plant hormone signal transduction, isoquinoline alkaloid biosynthesis, terpenoid backbone biosynthesis, lin- oleic acid metabolism. Among the 27 DEGs of plant hormone signal transduction, 5 DEGs of auxin-re- sponsive 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 geneCCD7 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 devel- opment of the micro-propagated plants.