Genome Sequence Of A Widely Cultivated Poplar And Its LncRNAs Response To Salt Stress

Poplars are widely cultivated in the world with ecologically and economically important value.Populus alba var.pyramidalis(=P.bolleana)is a variant of P.alba and has been widely planted due to its rapid growth,erect stems and high biomass production.In this study,we sequenced the genome,optimized the genetic transformation protocol and tried gene-knockouts of this variety.We further examined the spatio-temporal expression patterns of lncRNAs under salt stress.All these results provide an important basis for our understanding of evolutions of poplars and breeding new cultivars in the future.We optimized genetic transformation processes of P.alba var.pyramidalis.We established a highly efficient transformation protocol for this variety based on multiple experiments and reported protocols for other species.The average transformation efficiency of this variety was estimated to reach 23.6% by using this new protocol,and the average time from co-cultivation to whole plant regeneration was 80 days.Besides,we also established a transient transformation assay for this variety based on the leaves.Finally,we firstly established a CRISPR/Cas9-mediated gene knock-out system for this variety and the results showed a high knock-out efficiency of 89%.All of these protocols are highly useful to use this variety as a new model for diverse molecular and genetic studies.The genome sequence of P.alba var.pyramidalis was firstly reported.Through a hybrid genome assembly,a total of 460 Mb of the genome was assembled with the Illumina and Pacbio sequencing data.The genome has a scaffold N50 of 459 kb and a repeat sequence ratio of 44.61%.Annotation analyses predicted 37,901 protein-coding genes.This genome is highly collinear to that of P.trichocarpa,with most genes having orthologs between two species.We found a marked expansion of gene families related to the synthesis and transportation of auxin and cytokinin in P.alba var.pyramidalis.In particular,the histone HFO3 gene family has also expanded in the number of genes,which may be related to the higher genetic transformation efficiency of this variety.The NBS disease resistance gene family shows a significant contraction in number of genes in this variaty,likely due to its sensitivity to various pathogenic infections.The genome sequence presented here represents a valuable resource for further molecular functional analyses of this species as a new tree model,poplar breeding practices and comparative genomic analyses across different poplars.Long non-coding RNAs(lncRNAs)of P.alba var.pyramidalis were examined.We identified 10,531 lncRNAs in P.alba var.pyramidalis by using strand-specific transcriptome sequencing technology.The results showed that lncRNA has a high tissue specificity in expression.Tissue-specific expressed lncRNAs were found to participate in the process of plant growth regulation and environmental response.Under salt stress,about 60% of lncRNAs were differentially expressed.We systematically analyzed the spatio-temporal expression patterns of poplar lncRNAs in response to salt stress in P.alba var.pyramidalis and found that: 1)the differentially expressed lncRNAs have the high tissue specificity,and only 80 lncRNAs are differentially expressed in the four tissues;2)lncRNAs were mainly expressed under high salt stress;and 3)lncRNAs in different tissues regulated different target genes and participated in different functions.The response to salt stress was also different.We also found that lncRNAs can regulate many salt-resistant genes,such as the HKT1 and NHX genes associated with ion transport with high tissue specificity.The expression patterns of lncRNAs under salt stress recovered here laid a foundation for further understanding of the molecular adaptation and breeding new salt-and drought-tolerant cultivars.