Transcriptome Analyses And Population Genetics Of Psathyrostachys Huashanica

Psathyrostachys is a small genus consisting of less than ten species.It belongs to tribe Triticeae in family Poaceae,and distributes throughout the central Asia.Psathyrostachy huashanica is a cross-pollinated perennial herb in this genus.As an endemic species of China,P.huashanicais only geographically isolated distinct from all other and distributed only in mountain“Huashan”,which consists of rocks,with narrow area range from 350m to 2100m in altitude(Shaanxi province,central China).P.huashanica is an important gene pool applied to the improvement of wheat and grasses because of its valuable characteristics,such as early maturing,biotic and abiotic stress resistance and it is considered to be an important germplas m resource.Presently,it is a predominant species in its rigorous habitat with scattered distributio n and small population size.However,P.huashanica is listed as“National first class protected rare species”and“Crop wild relatives that are in urgent need of protection”,and is an example of species with increasing conservation interests in recent years.In previous studies,three key factors(e.g.the resistant ability of drought and salinity,habitat,or the process of populatio n’s development)might be the reasons for its endangerment,but the abiotic factor seems most seriously.However,due to the limited genomic information/lack of transcriptomic and the lack of key genetic markers,studies on adaptive evolution and speciation of P.huashanica face with a lot of difficulties.In this study,high-throughput comparative transcriptome sequencing was performed and generated large transcript sequences for functional characteristics and development of EST-SSR markers.Moreover,we explored genomic resources and provide insights into why and how P.huashanica could adapt to the harsh conditions characterized by cold,salinity or drought tolerance,as well as revealed the population structure of 149 individuals from 12 populations using the EST-SSR markers.Finally,118,173 unigenes were generated from approximately 99million reads after Ilumina pair-end sequencing.Differentially expressed genes revealed there are 11 candidate genes,which related to cold,salinity and drought tolerance based on the expression along altitude gradient.Meanwhile,the KEGG and GO annotation inferred that these candidate genes could have function in its environment adaptation.P.Huashanica seems to be more sensitive towards saline stress.From 118,173 unigenes of P.huashanica,13,261EST-SSR loci were detected and trinucleotide EST-SSRs were found to be the most abundant repeat motif.By designing the EST-SSR primers,110 pairs of primers have been selected randomly for further validation.Among them,11 pairs of primers were identified as polymorphic EST-SSR loci to further evaluate genetic diversity and population genetic structure.The results showed that 88 alleles were detected and the allele number for each locus ranged from 2 to 11.Moreover,the high genetic diversity(H_O=0.537±0.102,He=0.547±0.069,and PPL=91.37%)was found among 12 natural populations.Along the altitud ina l gradients,individuals from the 12 populations were mixed based on our Principal Coordinate Analysis and Bayesian population structure analyses,while two populations from high altitude were differentiated from others.Four of the collected populations were identified to have experienced genetic bottleneck during species evolutional history.The species with high genetic diversity is easy to adapt to the environmental changes.The bottleneck affected to the populations may inhibit the genetic differentiation.It is suggested that negative genetic consequences of habitat fragmentation may occur to some extent.However,previous study of P.huashanica showed decreasing genetic diversity with increasing altitude,which is confirmed to the hypothesis that this species has experienced genetic bottleneck found in current study.Furthermore,some upregulated/highly expressive genes in higher altitude but downregulated/non expressive in lower altitude were found.This genomic difference along altitude gradient may be caused by varying genes functions related with different altitudes.The identification of key stress responsive genes along altitude gradient and the low degree of divergence of higher altitude populations might clarify the occurrence and level of genetic differentiation in P.huashanica.In this study,we provide important insights into genes related to cold,drought and salinity tolerance involve in bad conditions.In point of conservation value,it is necessary to protect this important wild genetic resource to maintain its high level of genetic diversity through in-situ conservation.The molecular genetics resource identified in this study will facilitate further studies in functional genomics and population genetics in the Psathyrostachys species.The low degree of genetic differentiation along altitude gradient suggested that this endangered species is maybe at early stage of genetic differentiation and it may be developed into distinctive ecotype in future.In short,combined with transcriptome comparison,our study suggested the adaptation among different altitude in P.huashanicafrom Huashan Mountain.The adaptive differentiation may increase the genetic variations of populations along the altitudinal gradients.The identified candidate genes related to cold,salinity and drought tolerances,as well as the patterns of genetic differentiation of this species,may provide important genetic resources for breeding and species conservation in wild relatives.