Genome-Scale Transcriptome Analysis And Reference Genes Identification Of The Desert Shrub Artemisia Sphaerocephala

Artemisia sphaerocephala, a semi-shrub belonging to the Artemisia genus of the Compositae family, is an important pioneer plant that inhabits moving and semi-stable sand dunes in the deserts and steppes of northwest and north-central China. It is very resilient in extreme environments. Additionally, its seeds have excellent nutritional value, and the abundant lipids and polysaccharides in the seeds make this plant a potential valuable source of bio-energy. However, partly due to the scarcity of genetic information, the genetic mechanisms controlling the traits and environmental adaptation capacity of A. sphaerocephala are unknown. Here, we present the first in-depth transcriptomic analysis of A. sphaerocephala. To maximize the representation of conditional transcripts, m RNA was obtained from 17 samples, including living tissues of desert-growing A. sphaerocephala, seeds germinated in the laboratory, and calli subjected to no stress(control) and high and low temperature, high and low osmotic, and salt stresses. Additionally, in order to improve gene expression accuracy of the real-time PCR in A. sphaerocephala, based on transcriptome data, we selected 21 candidate reference genes to do the real-time PCR analysis in vegetative period, reproductive stage, seed development stage, under abiotic conditions and all samples of A. sphaerocephala. And then, Ge Norm, Norm Finder and Best Keeper were used to calculate stable value of candidate reference genes. There are several main results as following:1. De novo transcriptome assembly performed using an Illumina Hi Seq 2500 platform resulted in the generation of 46,831,604 reads(9.46 Gb). All high-quality clean reads were assembled de novo with Trinity software, which generated 137,060 transcripts. After further analyses were performed, 68,373 unigenes with a mean length of 692.76 nt and an N50 of 1,161 nt were obtained. And out of the 68,373 unigenes, 40,153(58.70%) significantly matched sequences deposited in the public protein databases.2. We analyzed the key genes involved in the unsaturated fatty acid synthesis pathway and identified 26 fad2, 3 fad3, 1 fad6, and 9 fad7/8 genes 26 in A. sphaerocephala, and we constructed phylogenetic tree of fad2, which is the largest fad2 gene family reported to date.3. A set of genes responsible for resistance to extreme temperatures, salt, drought and a combination of stresses was identified. That showed adaptive mechanism in A. sphaerocephala may be associated with the regulation function of these genes.4. According to Ge Norm, Norm Finder and Best Keeper results, in A. sphaerocephala, UBC9 and Actin can be considered as suitable reference genes in vegetative period; UBC9 and PTB can be used as suitable reference genes in reproductive stage; UBC and PTB can be used as suitable reference genes in seed development stage; Clathrin adaptor complex subunit and Expressed 4 can be used as suitable reference genes under abiotic conditions; UBC9 and TIP41-like can be considered as suitable reference genes in all samples.The present work provides abundant genomic information for functional dissection of the important traits of A. sphaerocephala and contributes to the current understanding of molecular adaptive mechanisms of A. sphaerocephala in the desert environment. Identification of the key genes in the unsaturated fatty acid synthesis pathway could increase understanding of the biological regulatory mechanisms of fatty acid composition traits in plants and facilitate genetic manipulation of the fatty acid composition of oil crops.