Background: Ramie fiber, extracted from vegetative organ stem bast, is one of the most important natural fibers. Understanding the molecular mechanisms of the vegetative growth of the ramie and the formation and development of bast fiber is essential for improving the yield and quality of the ramie fiber. However, only 418 expressed tag sequences (ESTs) of ramie deposited in public databases are far from sufficient to understand the molecular mechanisms. Thus, high-throughput transcriptome sequencing is essential to generate enormous ramie transcript sequences for the purpose of gene discovery, especially genes such as the cellulose synthase (CesA) gene. Results: Using Illumina paired-end sequencing, about 53 million sequencing reads were generated. De novo assembly yielded 43,990 unigenes with an average length of 824 bp. By sequence similarity searching for known proteins, a total of 34,192 (77.7%) genes were annotated for their function. Out of these annotated unigenes, 16,050 and 13,042 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 19,846 unigenes were mapped to 126 KEGG pathways, and 565 genes were assigned to http://starch and sucrose metabolic pathway which was related with cellulose biosynthesis. Additionally, 51 CesA genes involved in cellulose biosynthesis were identified. Analysis of tissue-specific expression pattern of the 51 CesA genes revealed that there were 36 genes with a relatively high expression levels in the stem bark, which suggests that they are most likely responsible for the biosynthesis of bast fiber. Conclusion: To the best of our knowledge, this study is the first to characterize the ramie transcriptome and the substantial amount of transcripts obtained will accelerate the understanding of the ramie vegetative growth and development mechanism. Moreover, discovery of the 36 CesA genes with relatively high expression levels in the stem bark will present an opportunity to understand the ramie bast fiber formation and development mechanisms.

Drought is the main environmental factor impairing hemp growth and yield. In order to decipher the molecular responses of hemp to drought stress, transcriptome changes of drought-stressed hemp (DS1 and DS2), compared to well-watered control hemp (CK1 and CK2), were studied with RNA-Seq technology. RNA-Seq generated 9.83, 11.30, 11.66, and 11.31 M clean reads in the CK1, CK2, DS1, and DS2 libraries, respectively. A total of 1292 differentially expressed genes (DEGs), including 409 (31.66%) upregulated and 883 (68.34%) downregulated genes, were identified. The expression patterns of 12 selected genes were validated by qRT-PCR, and the results were accordant with Illumina analysis. Gene Ontology (GO) and KEGG analysis illuminated particular important biological processes and pathways, which enriched many candidate genes such as NAC, B3, peroxidase, expansin, and inositol oxygenase that may play important roles in hemp tolerance to drought. Eleven KEGG pathways were significantly influenced, the most influenced being the plant hormone signal transduction pathway with 15 differentially expressed genes. A similar expression pattern of genes involved in the abscisic acid (ABA) pathway under drought, and ABA induction, suggested that ABA is important in the drought stress response of hemp. These findings provide useful insights into the drought stress regulatory mechanism in hemp.

Ramie fiber extracted from stem bark is one of the most important natural fibers. Drought is a main environment stress which severely inhibits the stem growth of ramie and leads to a decrease of the fiber yield. The drought stress-regulatory mechanism of ramie is poorly understood.

Quantitative real-time PCR (qPCR) is commonly used for deciphering gene functions. For effective qPCR analyses, suitable reference genes are needed for normalization. The objective of this study is to identify the appropriate reference gene(s) for qPCR analyses of the leaves and roots of ramie (Boehmeria nivea L.), an important natural fiber crop. To accomplish this goal, we investigated the expression patterns of eight common plant qPCR reference genes in ramie leaves and roots under five abiotic stresses, five hormonal treatments, and one biotic stress. The relative expression stabilities of the eight genes were evaluated using four common but different approaches: geNorm, NormFinder, BestKeeper, and RefFinder. Across the 11 tested conditions, ACT1 was the most stably expressed among the eight genes while GAPDH displayed the biggest variation. Overall, while variations in the suggested reference genes were found for different tissue x treatment combinations, our analyses revealed that together, genes ACT1, CYP2, and UBQ can provide robust references for gene expression studies of ramie leaves under most conditions, while genes EF-1α, TUB, and ACT1 can be used for similar studies of ramie roots. Our results should help future functional studies of the genes in ramie genome across tissues and environmental conditions.

Ramie (Boehmeria nivea L. Gaud) is one of the most important natural fiber crops, and improvement of fiber yield and quality is the main goal in efforts to breed superior cultivars. However, efforts aimed at enhancing the understanding of ramie genetics and developing more effective breeding strategies have been hampered by the shortage of simple sequence repeat (SSR) markers. In our previous study, we had assembled de novo 43,990 expressed sequence tags (ESTs). In the present study, we searched these previously assembled ESTs for SSRs and identified 1,685 ESTs (3.83%) containing 1,878 SSRs. Next, we designed 1,827 primer pairs complementary to regions flanking these SSRs, and these regions were designated as SSR markers. Among these markers, dinucleotide and trinucleotide repeat motifs were the most abundant types (36.4% and 36.3%, respectively), whereas tetranucleotide, pentanucleotide, and hexanucleotide motifs represented <10% of the markers. The motif AG/CT was the most abundant, accounting for 28.74% of the markers. One hundred EST-SSR markers (97 SSRs located in genes encoding transcription factors and 3 SSRs in genes encoding cellulose synthases) were amplified using polymerase chain reaction for detecting 24 ramie varieties. Of these 100 markers, 98 markers were successfully amplified and 81 markers were polymorphic, with 2–6 alleles among the 24 varieties. Analysis of the genetic diversity of all 24 varieties revealed similarity coefficients that ranged from 0.51 to 0.80. The EST-SSRs developed in this study represent the first large-scale development of SSR markers for ramie. These SSR markers could be used for development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping, and cultivar fingerprinting.