Selection Of Reference Genes,and Gene Mining And Expression Pattern Analysis Of NAC And HSP20 Gene Family In Switchgrass

Switchgrass(Panicum virgatum L.),as a warm season C4 perennial grass that is native to North America,is regarded as an important biofuel crop for its remarkable biomass yield and good adaptability.In order to protect the food safety,swictchgrass are usually plant in marginal field suffered from different abiotic stresses,which severely influence the yield and quality of switchgrass.Therefore,it is significant to focus on switchgrass under different abiotic stresses.In this study,14 candidate reference genes in switchgrass were evaludated under various abiotic stresses,and the most stable reference genes were selected out for different stresses,and we also made a comprehensive analysis for NAC and Hsp20 gene family members,and explored many genes related to stress and growth of switchgrass.The main research results were as follows:1.Screen out the reference genes of switchgrass under abiotic stressesQuantitative real-time reverse transcriptase PCR(qRT-PCR)is a sensitive and powerful technique for measuring differential gene expression;however,changes in gene expression induced by abiotic stresses are complex and multifaceted.Therefore,a set of stably expressed reference genes for data normalization is required.Switchgrass is a prime candidate crop for bioenergy production.The expression stability of reference genes in switchgrass,especially under different experimental conditions,is largely unknown.In order to identify the most suitable reference genes for abiotic stress studies in switchgrass,we evaluated 14 candidate genes for their expression stability under drought,high salinity,cold,heat,and waterlogging treatments using the Delta Ct,geNorm,BestKeeper,and NormFinder approaches.Validation of reference genes indicated that the best reference genes should be selected based on the stress treatment.Actin 2(ACT2),Carotenoid-binding protein 20(CBP20),and Tubulin(TUB)were found to have the highest expression stability to study drought stress.18S ribosomal RNA1(18S rRNA1),ACT2,and TUB were the most stably expressed genes under salt stress.Ubiquitin-conjugating enzyme(UBC),TUB,and Cyclophilin2(CYP2)were the most suitable reference genes across cold treatments.Likewise,18S rRNA1,UBC,and TUB were good reference genes for studying heat stress,while ACT2,18S rRNAl,and ubiquitin3(UBQ3)were the top three reference genes under waterlogging treatment.Considering that reference gene expression may vary across switchgrass tissues,ACT2 and 18S ribosomal RNA2(18S rRNA2)were shown to be the most stably expressed genes in switchgrass leaves and roots,respectively.The highly ranked reference genes that were identified in this study were shown to be capable of detecting subtle differences in the expression rates of other genes.These differences may have beenmissed if a less suitable reference gene was used.2.Comprehensive analysis of NAC family members in switchgrassNAC proteins comprise of a plant-specific transcription factor(TF)family and play important roles in plant development and stress responses.Switchgrass is the prime candidate and model bioenergy grass across the world.Excavating agronomically valuable genes is important for switchgrass molecular breeding.In this study,a total of 251 switchgrass NAC(PvNACs)family genes clustered into 19 subgroups were analyzed,and those potentially involved in stress response or tissue-specific expression patterns were pinpointed.Specifically,27 PvNACs were considered as abiotic stress-related including four membrane-associated ones,and 40 PvNACs with tissue-specific expression patterns were found among that eight might involve in lignin biosynthesis and/or secondary cell wall formation.Conserved functional domains and motifs were also identified among the PvNACs and potential association between these motifs and their predicted functions were hypothetically proposed,that together shed an insight on further experimental studies on PvNACs and genetic improvement of switchgrass in the future.3.Comprehensive analysis of Hsp20 family members in switchgrassHsp20 proteins exist in all plant species and represent the most abundant small heat shock proteins(sHSPs)in plants.Hsp20s were known as chaperones maintaining cellular homeostasis during heat or other kinds of abiotic stresses.The objective of this study was to understand the phylogenetic relationship,genomic organization,diversification of motif modules,genome localization,expression profiles,and interaction networks of switchgrass Hsp20s(PvHsp20s).A total of 63 PvHsp20s were identified with their consensus as well as unique ACD motifs and gene structures analyzed.Most PvHsp20s(87%)were responsive to heat and other kinds of abiotic stresses.When under optimum growth condition,38 of them displayed relative higher expression levels in inflorescence and seeds,suggesting their protective roles in the stress-sensitive reproductive organs.An in silico analysis of interaction network of PvHsp20 proteins further revealed potential interactive proteins,including stress-inducible ones in the network.Furthermore,PvHsp20 genes unevenly distributed in two sets of homeologous chromosomes,and only segmental duplication was found among the paralogous gene pairs,reflecting that the allotetraploidization of switchgrass allowed the accumulation of PvHsp20s that in turn facilitated its successful adaptation in hot and dry plateaus of North America.The present results provided an insight into PvHsp20s with an emphasis on the uniqueness of this gene family in switchgrass.Such information shall also be useful in functional studies of PvHsp20 genes and molecular breeding of switchgrass.