Transcriptomics Studies On Homoploid Hybridization And Adaptive Evolution In Hippophae(Elaeagnaceae)

Natural hybridization is a hybridization event occurred between different taxon under natural conditions,and of great significance to biological evolution and speciation.With the advancement of genome sequencing technology,people began to pay attention to the relationship between genes expression and its ecological role during speciation at the omics level,and it also provided an opportunity to explore the ecological genetic mechanism in process of hybrid origin.In this paper,individuals of Hippophae goniocarpa Lian and its parental species,H.rhamnoides ssp.sinensis and H.neurocarpa,all dwelling on the hybrid zone in Rixucun of Qilian,Qinghai province,were collected as well as individuals from their exotically distributed populations in the same county.The next generation sequencing of Hippophae RNAseq was performed on the Illumina Hi Seq 2500 platform.The third-generation sequencing of transcriptome was performed using the Pac Bio RS II platform only for the No.4 individual of H.goniocarpa.The purpose of this transcriptomics study was to confirm the hybrid origin of H.goniocarpa in this genus,and further dissect the genetic composition of the hybrids,as well as the molecular basis of the adaptive differentiation between H.goniocarpa,H.rhamnoides ssp.sinensis and H.neurocarpa.The main results are listed as the following:(1)Illumina high-throughput sequencing of three sea buckthorn produced total clear bases 253.76G,then unigenes 199145 with N50 ranging from 792 bp to 1247 bp.Unigenes were annotated by the seven databases,NR,NT,GO,Swiss Prot and so on.There were 132,409 genes annotated at least by one of the seven databases,accounting for 66.48%of all unigenes.The maximum length of the full-length transcripts of Pac Bio was 17,026 bp with N50 of 2405 bp.The third-generation sequencing technology of full-length transcriptome showed great advantages in reads length.Then,Sanger sequencing technology was used to evaluate the accuracy of the two high-throughput sequencing technologies in this study.It was found to be about99.54%,just greater than the reported 99.14%in the next generation technology,and to reach 99.14%in the third-generation results,far greater than the generally accepted82.10%-84.90%,because the full-length data was calibrated using the next generation sequencing data in this study.(2)The phylogenetic trees were constructed using 171 ortholog groups(OG)obtained from the next generation sequencing data.Their topological structure could be divided into H.rhamnoides ssp.sinensis S branch and H.neurocarpa N branch.After adding the full-length data,the two allelic copies among homologs in the H.goniocarpa No.4 individual were clustered into the S branch and the N branch respectively.The above two alleles were cloned and sequenced in three species,and each of them could be only found in H.rhamnoides ssp.sinensis and H.neurocarpa respectively,showing both of them had acted as parents of H.goniocarpa during its hybrid origin.All above"Omics"data further proved that the H.goniocarpa originated from the natural hybridization between H.rhamnoides ssp.sinensis and H.neurocarpa.On the other hand,the Pac Bio results of the single molecules sequencing data shows very powerful and rather suitable for identifying the homoploid hybridization speciation.(3)In comparative analysis of the transcriptome data,60773,60660,60688 and60770 heterozygous sites among the 398,063 SNPs were respectively detected in the four individuals of H.goniocarpa in Rixucun that showed the additivity of the species-specific SNP of H.rhamnoides ssp.sinensis and H.neurocarpa in the nuclear genome sequence.Further analysis showed that the genes containing single signals of gene conversion in the four individuals of H.goniocarpa were 409,478,479 and 419,respectively,and the genes containing multiple signals of gene conversion were 146,145,156 and 136,respectively.The haplotype block with multiple conversion sites ranged from 3 bp to 7639 bp in length,and the SNP sites converting to H.rhamnoides ssp.sinensis is 2-3 times than that to H.neurocarpa.The above results indicate that the individuals of H.goniocarpa in Rixucun should have experienced a stage of sexual reproduction including meiosis just after its hybrid origin,and it is speculated that at least one generation of self-crossing of the F₁hybrid or its backcrossing to the parents had taken place sometime.(4)The differential analysis was used to distinguish the gene expressions among H.neurocarpa(N?R),H.rhamnoides ssp.sinensis(S?R)and H.goniocarpa(G?R)in the Rixucun hybrid zone.It was found that H.goniocarpa(S?G)up-regulated 1357genes and down-regulated 1743 genes in comparison with H.rhamnoides ssp.sinensis(S?R).But the genes for up-regulation and down-regulation in H.goniocarpa(G?R)were 865 and 76 when compared it to H.neurocarpa,respectively.While,the up-regulated and down-regulated genes of H.goniocarpa(G?R)were 553 and 86 to H.rhamnoides ssp.sinensis,respectively.The hybrid species H.goniocarpa had more up-regulated expression and less down-regulated genes than the two parental species.The up-regulated expression genes were mainly related to secondary metabolite synthesis,hormone response,photosynthesis and immune resistance pathway,showing a certain heterosis in the hybrid species.In the 171 orthologous gene cluster OG of three sea buckthorn species,17 orthologous gene clusters containing signals of Darwin positive selection were found,the majority of their gene function were related to cold resistance,protein phosphorylase and DNA repair.The above results indicate that three sea buckthorns had suffering from adaptive differentiation at both levels of gene expression and gene function.