Genetic Diversity and Transcriptomic Analysis for Salt Tolerance in Miscanthus

Abstract

Miscanthus is a potential bioenergy crop due to its C4 perennial growth, outstanding productivity, high water and nutrient use efficiency, and high abiotic stress tolerance, but has not been much studied in Korea although Korea has diverse genetic resources of Miscanthus species. Abiotic stress tolerance in a potential bioenergy crop is an important trait for its commercial cultivation in marginal lands, but little information is available concerning abiotic stress tolerance, particularly salt stress tolerance, in Miscanthus. Therefore, this study was conducted to evaluate genetic diversity of Miscanthus accessions collected in Korea and its neighboring countries, and to investigate physiological and molecular responses of Miscanthus to salt stress. Thus, a series of studies including SSR marker analysis, salt-response study, and leaf transcriptome analysis were conducted. Ploidy estimation and SSR analysis using 24 selected SSR markers of 286 Miscanthus accessions collected mainly in Korea and its neighboring countries revealed that they are classified into two main taxonomic groups, sections Triarrhena and Miscanthus, and showed high genetic diversity. The first group was mainly composed with M. sacchariflorus and divided into sub-groups depending on their ploidy levels (2n=2x or 4x). M. × giganteus and M. lutarioriparius were closely grouped with M. sacchariflorus and the second group consisted of M. sinensis and M. floridulus, suggesting that the combined analysis using ploidy estimation and SSRs analysis with the selected markers is effective for classification and diversity assessment of Miscanthus species. Whole plant assay at a range of salt concentrations up to 200 mM showed that physiologic parameters such as chlorophyll contents, photosynthesis, stomatal conductance and plant growth responded to salt stress in both M. sinensis and M. sacchariflorus. Interspecific difference in plant response was observed at the above 100 mM salt condition with photosynthesis rate being the most sensitive parameter. The estimated GR50 values in shoot dry weight were 74.96 mM and 52.57 mM for M. sinensis and M. sacchariflorus, respectively, implying that M. sinensis is more tolerant to salt stress than M. sacchariflorus. Further, a total of 35 Miscanthus accessions consisting of 21 M. sinensis, 13 M. sacchariflorus, and a M. × giganteus were tested their survival and growth capacities at 60 and 100 mM salt condition and showed intraspecific diversity in adaptability under salt stress. The accessions of M. sinensis had broader intraspecific variation than M. sacchariflorus for salt adaptability. Through this experiment, the most salt-sensitive accession (M-119) and the most salt-tolerant one (M-131) were selected. . Transcriptome analysis of two M. sinensis accessions M-119 (salt-sensitive) and M-131 (salt-tolerant) leaves sampled at 0, 24, 48 hours after salt treatment using Illumina sequencing platform resulted in a total of 363 DEGs obtained from the pair-wise comparisons. For M-131, 178 DEGs were significantly up-regulated while 118 DEGs were down-regulated during the first 24 hours after salt treatment, then many of DEGs were regulated in the reverse direction during subsequent 24 hours. Whereas for M-119, 143 genes were up-regulated and 170 genes were down-regulated in 24 hours after salt treatment, and the regulated direction of DEGs was almost maintained up to 48 hours after treatment. The up-regulated DEGs during 24 hours showed differences in the numbers and the kinds of DEGs, and even in the expression patterns between the two accessions during time-course. These differences between the two M. sinensis accessions might be led by their intrinsic abilities such as responsiveness of signal perception and efficiency of signal transduction in plant salt-tolerance cascades. Six TFs and ten RLKs, as previously known abiotic stress responsible genes, were exclusively up-regulated in M-131 during the first 24 hours after salt treatment. Thus, our results suggest that gene expression regulations of the several upstream genes in the salt-tolerance cascades brought about adaptive diversity in M. sinensis.