Comparative Plastomic Studies Of Dendrobium Species And Comparison Of The Physiological Effects And Transcriptome Responses Of Dendrobium Officinale Under Different Abiotic Stresses

Orchidaceae(common name:orchids)is the largest family in monocots,comprising about 25,000 species in 880 genera and five subfamilies.Many orchids are of great values because of their beautiful and long-lasting flowers.However,phylogenetic relationships among the five orchid subfamilies remain unresolved.The major dispute centered on whether the three one-stamened subfamilies,Epidendroideae,Orchidoideae,and Vanilloideae,are monophyletic or paraphyletic.Moreover,the plastome structural changes and effective genetic loci at the species-level phylogenetics of orchids have been rarely documented.In this study,we compared 53 orchid plastomes,including four newly sequenced ones that represent four remote genera:Dendrobium,Goodyera,Paphiopedilum,and Vanilla.These four plastomes differ from one another not only in their lengths of inverted repeats(IRs)and small single copy(SSC)regions but also in the retention of ndh genes.Comparative analyses of the orchid plastomes revealed that the expansion of IRs in Paphiopedilum and Vanilla is associated with loss of ndh genes.In orchid plastomes,mutational hotspots are genus-specific.With carefully estimated,we proposed that the three loci:5 ’trnK-rps16,trnS-trnG and rpsl6-trnQ could be a powerful marker for the genera within Epidendroideae,and clpP-psbB,rpsl6-trnQ for Cypripedioideae.After analysis of partitioned dataset,our plastid phylogenomic trees were congruent in topologies,in which two one-stamened subfamilies(i.e.,Epidendroideae and Orchidoideae)were placed as sister to a multi-stamened subfamily(i.e.,Cypripedioideae)rather than to the other one-stamened subfamily(Vanilloideae),suggesting that the living one-stamened orchids be paraphyletic.Moreover,the complete plastome sequences of Apostasia wallichii and Neuwiedia zollingeri var.singapureana were sequenced and compared with 43 previously published photosynthetic orchid plastomes to characterize the plastome structure and evolution in the orchids.Unlike many orchid plastomes(e.g.,Paphiopedilum and Vanilla),the plastomes of Apostasioideae contain a full set of 11 functional ndh genes.The distribution of repeat sequences and SSR elements enhance the view that the evolution rate of non-coding regions was higher than that of coding regions.The ten loci-ndhA intron,matK-5’trnK,clpP-psbB,rps8-rpl14,trnT-trnL,3’trnK-matK,clpP intron,psbK-tnK,trnS-psbC and ndhF-rpl32-that had the highest degrees of sequence variability(SV)were identified as mutational hotspots for the Apostasia plastome.Furthermore,plastid genes exhibit a variable evolution rate in one orchid specie and among different genera;non-coding regions evolve diversified among orchid species.Those results suggest that there is a disproportional plastome-wide variation of substitution rates among orchid species.Additionally,the sequences flanking the IR/SSC junctions of photosynthetic orchid plastomes were categorized into three types according to the presence/absence of ndh genes.Different evolutionary dynamics for each of the three IR/SSC types of photosynthetic orchid plastomes are also proposed.Dendrobium is one of the largest genera in Orchidaceae,comprising about 1600 species mainly distributed in tropical Asia,Australasia,and Australia.There are 74 species and two varieties of this genus in China.Because of their ornamental and commercial value,Dendrobium orchids have been studied at low taxonomic levels.However,structural changes and effective mutational hotspots of Dendrobium plastomes have rarely been documented.Here,30 Dendrobium plastomes were compared,comprising 25 newly sequenced in this study and five previously published.Except for their differences in ndh genes,these plastomes shared identical gene content and order.Comparative analyses revealed that the variation in size of Dendroubium plastomes was associated with dramatically changed length of InDels.Furthermore,ten loci were identified as the top-ten mutational hotspots,whose sequence variability was almost unchanged with more than 10 plastomes sampled,suggesting that they may be powerful markers for Dendrobium species.Moreover,primer pairs of 47 polymorphic microsatellites were developed.After assessing the mean BS values of all combinations derived from the top-ten hotspots,we recommend that the combination of five hotspots-trnT-trnL,rpl32-trnL,clpP-psbB,trnL intron,and rps16-trnQ—should be used in the phylogenetic and identification studies of Dendrobium.Furthermore,on the base of the comprehensive comparison of the medicinal Dendrobium plastome,we surmised four most informative hotspot regions,which can be used for the authentication study.Additionally,the impact of three evolutionary forces-selection,mutational biases,and GC-biased gene conversion(gBGC)-on the variation of GC content in Dendrobium plastomes was evaluated.Our results revealed:(1)consistent GC content evolution trends and mutational biases in single-copy(SC)and inverted repeats(IRs)regions;and(2)that gBGC has influenced the plastome-wide GC content evolution.These results suggest that both mutational biases and gBGC affect GC content in the plastomes of Dendrobium genus.In orchids,the plastid ndh genes have experienced independent loss among different orchid genera.Plastid ndh proteins comprise a complex that joins the cyclic electron transport(CET)at photosystem I,which involved in the response to abiotic stress.Dendrobium officinale is facultative CAM plant.In response to different abiotic stress,the photosynthetic carbon cycle of Dendrobium officinale will switch between C3 to CAM pathway.To compare the responses to multiple stresses,we compared physiological effects and transcriptome changes in Dendrobium officinale under four abiotic stresses(low light stress,high light stress,cold stress and drought stress).Our results revealed the antioxidant enzymes activities differed among four abiotic stresses.The increased content of proline in different abiotic stresses suggests that the proline plays an important role in responses to multiple stresses.Moreover,the transcriptome data indicated that the biosynthesis of proline was through the Glu-pathyway under abiotic stresses.According to the physiological characteristics of photosynthesis and transcriptome data,the photosynthesis of endrobiumw offcinale was changed in different abiotic stresses,especially for drought stress.Moreover,the PET pathway was changed significantly among photosynthesis.Additionally,the variable expression of plant hormone related genes and transcript factors will provides new insights to improve our understanding of the mechanisms of Dendrobium officinale under different abiotic stress in the further studies.