Transcriptomics Study On Natural Hybridization Of Hippophae Neurocarpa And H.thibetana

With the continuous development of high-throughput transcriptome sequencing technology,RNA-seq technology has become an important research method in the fields of gene expression research and transcriptome analysis,and it has been widely used in plant studies.Natural hybridization plays an extremely important role in the evolution of plant species and is a hotspot in systems and evolutionary biology.Generally,the identification of natural hybrids is mainly through morphological observation and molecular biological methods such as nuclear gene and cytoplasmic gene phylogeny.In recent years,more and more researchers have chosen to use transcriptomics methods to study speciation and evolution.Hippophae L.are mainly distributed in the Qinghai-Tibet Plateau and its marginal high-altitude areas,and have important ecological and economic value.Studies have shown that natural hybridization occurs frequently between plant species of this genus.In the field investigation,the laboratory finds that there may be a hybridization event between Hippophae neurocarpa S.W.Liu et T.N.He and Hippophae thibetana Schlechtend..Therefore,this study uses H.neurocarpa,H.thibetana and their putative hybrid offspring(H.neurocarpa × H.thibetana)as the experimental subjects.Through the second-generation transcriptome sequencing of the Illumina Hi Seq sequencing platform and the third-generation Pacific Biosciences RS sequencing platform,the method of transcriptomics is used to study the natural hybridization between H.neurocarpa and H.thibetana and the genetic composition of the hybrid offspring,providing evidence for the hybridization and speciation of Hippophae L.plants.The main results are as follows:(1)Through total RNA extraction,cDNA library construction,Illumina Hi Seq high-throughput sequencing,filtering low-quality data and transcriptome splicing,a large amount of transcriptome datas are obtained.The length of Unigenes is the largest in the 200bp-500 bp interval,and the N50 is between 512 bp and 870 bp.The function of Unigenes in the seven databases of NR,NT,PFAM,Swiss Prot,GO,KOG,and KO are annotated.It is assumed that the length of the third-generation transcriptome sequencing transcripts of the putative hybrid offspring is the largest in the range of1k-2kbp.The average sequence length is 2 212 bp,and the N50 is 2 518 bp,which is much larger than the N50 of the second-generation sequencing.This indicates that the third generation transcriptome sequencing has great advantages in the aspect of sequence reading length.In addition,the Sanger method is used to verify the accuracy of the second and third generation transcriptome sequencing results.The results show that the accuracy of the second and third generation transcriptome sequencing is high,and the data obtained could be used later.(2)Ortho Finder software is used to screen Single Copy Orthogroups,a total of 5182 groups,of which a total of 147 groups have a positive selection effect,these genes play an important role in the evolution of H.neurocarpa,H.thibetana and H.neurocarpa × H.thibetana.(3)Through the analysis of a large number of orthologous gene sequences,it is found that the putative hybrid offspring in the Dari area shows the additivity of the different nucleotide sites of the two parents;In the phylogenetic tree constructed by using orthologous genes,H.neurocarpa clusters into one branch,H.thibetana clusters into one branch,and the putative hybrid progeny gathers into the branches of H.neurocarpa and H.thibetana in different forms,showing the characteristics of phylogenetic relationship of the hybrid progeny.At the level of transcriptomics,it proves that there is a natural hybridization between H.neurocarpa and H.thibetana.(4)The results of transcriptome sequence analysis show that the putative hybrid offspring with morphological characteristics of the two parents found in the field survey in Dalang Village,Qinghai Province,does not have the additivity of the gene sequence of H.neurocarpa and H.thibetana,but is similar to the gene sequence of H.thibetana;The phylogenetic trees constructed based on orthologous genes are clustered into a branch with H.thibetana,which proves that it is not a hybrid offspring of H.neurocarpa and H.thibetana,but should be H.thibetana.(5)There are gene fragments and gene locus gene conversion events in the hybrid offspring individuals of Dari region to the parent H.neurocarpa and/or H.thibetana,indicating that the hybrid offspring formed by natural hybridization between H.neurocarpa and H.thibetana is not the F1 generation,but may have undergone the stage of sexual reproduction based on meiosis.