De Novo Genome Sequencing And Transcriptome Sequencing To Resolve The Molecular Basis Of Yinshania Henryi Tolerance To High Calcium Stress

Karst landform is widely distributed worldwide,and in China they are mainly found in the southwest.It is an environment characterized by high soil calcium and magnesium content,low light intensity,and shallow soil layers with poor water holding capacity,and this special environmental selection pressure has given plants in this region specific environmental adaptability.However,few studies have been conducted on the ability of plants to adapt to karst high calcium environment at the genomic level.Yinshania henryi is a Brassicaceae plant that can adapt to high calcium karst environments.To explore the molecular basis of Y.henryi adaptation to the karst environment,this study first used the three-generation sequencing and Hi-C technology to obtain a high-quality genome.On this basis,the role of genome evolution in adapting Y.henryi to the karst environment dominated by high calcium was explored.In addition,transcriptome sequencing was performed on Y.henryi treated with different concentrations of Ca²⁺（1.5,10,25,50,and 100 m M）to explore the Ca²⁺transport and the expression of genes related to the response to Ca²⁺treatment.To reveal the molecular basis of Y.henryi tolerance to karst high calcium environment.The main results of this study are as follows:1.The primary assembly of the genome of Y.henryi was performed using three-generation sequencing data obtained from the Pacbio Seque II platform,and the resulting genome draft consisted of 274 contigs with a size of 202.13 Mb and a contig N50 of 16.03 Mb.The genome of Y.henryi was also anchored to the chromosome level using Hi-C technology,and the total length of the six chromosomes was 188.92 Mb,accounting for 93.46%of the genome draft length,and the scaffold N50 length was 34.35 Mb.The genome integrity assessment showed that 97.6%of the BUSCO（1,614）gene set conserved in embryonic plants could be completely compared to the individual genes of the Y.henryi genome,indicating that the genome The integrity of the genome was good.The total length of repeat sequences annotated in the Y.henryi genome was 88.70 Mb,accounting for 43.89%of the genome size;the number of genes predicted in Y.henryi genome was28,473,of which 27,947 genes were annotated to function.2.With reference to the existing phylogenetic relationships of the Brassicaceae,the genomes of 14 Brassicaceae species were downloaded from public databases and subjected to comparative genomic analysis with Y.henryi.The results of the phylogenetic analysis showed that Y.henryi belongs to the LI lineage of the existing 4 lineages of the Brassicaceae,which is consistent with the available results.Gene family expansion contraction analysis showed that a total of 87 gene families of Y.henryi underwent significant expansion（P<0.05）,and these genes were mainly enriched in pathways related to redox,energy metabolism,and carbohydrate metabolism.It is thought to be related to the better tolerance of Y.henryi to karstic high calcium environment.3.Y.henryi leaves treated with different Ca²⁺concentrations were subjected to transcriptome sequencing and screened for differentially expressed genes.The results showed that high calcium treatment（100 m M）increased the expression of several CML genes with calcium signal decoding ability,which could presumably improve the tolerance of Y.henryi to high calcium environment by participating in decoding calcium signals in Y.henryi cells under high calcium environment.In addition,high calcium treatment increased the expression of calcium transporter-related genes（CAX,ACA,and CNGC）.These genes could reduce the damage caused by high calcium environment to plants by translocating excess Ca²⁺from the cytoplasm to the vacuole or outside the cells.4.Enrichment analysis of differentially expressed genes showed that high Ca²⁺treatment（100 m M）induced upregulation of the expression of genes related to ABA（abscisic acid）synthesis in the carotenoid biosynthetic pathway and increased ABA accumulation in Y.henryi.At the same time,ABA accumulation activated Sn RK2,a core component of the ABA signaling pathway,and two Sn RK2 gene expression was upregulated in high Ca²⁺treatment.Therefore,it is believed that the up-regulation of the expression of genes related to ABA synthesis and its signaling pathway enhanced Y.henryi tolerance to high Ca²⁺stress.In addition,genes encoding glutathione S-transferase,peroxiredoxin-6,glutathione peroxidase,and glutathione reductase in the glutathione metabolic pathway were differentially expressed under high Ca²⁺treatment.These genes could enhance the tolerance of Y.henryi to high Ca²⁺treatment by scavenging reactive oxygen species generated by the process of high Ca²⁺treatment.5.To ensure the accuracy of the transcriptome data,15 RNA samples extracted for the construction of the sequencing library were used,and some 19 important genes related to decoding calcium signal,Ca²⁺transport and response to Ca²⁺treatment were selected and validated by real-time fluorescence quantitative PCR.The results showed that under high Ca²⁺treatment,the expression of calcium decoding genes,mainly CML19/37/40,and Ca²⁺transport genes,mainly CAX1/3,ACA8/10/13 and CNGC2/20,were significantly up-regulated,similar to the trend of transcriptome data;meanwhile,the expression of transcription factors（WRKY6）,flavonol synthase（FLS1）,chlorophyllase（CLH1）,and aquaporin（PIP2-5）genes associated with the response to Ca²⁺treatment also showed changes.This result indicates that these genes play an important role in the tolerance of Y.henryi to high Ca²⁺stress.In summary,this study demonstrates that Y.henryi,in a high calcium environment,can decode calcium signals through CML,activate transcription factors and ABA signaling pathways,regulate the expression of CAX,ACA,CNGC genes and genes in the glutathione metabolic pathway,sequester or exclude excess Ca²⁺from the cytoplasm,and scavenge reactive oxygen species（ROS）accumulated by the stress process,as well as regulate the expression of some other important genes related to the response to high Ca²⁺stress,so that it can maintain its own normal metabolic process.