The Analysis Of Developmental Mechanisms Of Cones Of Pinus Massoniana And Its Introgression Hybrid At Transcriptome Level

Pinus massoniana and P.hwangshanensis are two conifer species mainly distribute in southern China,which are of economic and ornamental value,respectively.Around the middle and lower reaches of the Yangtze River,P.massoniana occurs mainly at altitudes below 700 m,while P.hwangshanensis can be found above 900 m.At altitudes where the distribution of both pines overlaps,a natural introgression hybrid exists,which we will further refer to as the ‘Z pine'.This pine has a morphological character that shares attributes of both P.massoniana and P.hwangshanensis.However,compared to other two pines,its reproductive structure,the pinecone,has an ultra-low ripening rate with seeds that germinate poorly.In this study,we aimed to find the reason for the impaired cone maturation by comparing transcriptome libraries of P.massoniana and Z pine cones at seven successive growth stages,looking for crucial differentially expressed genes(DEGs),GO enrichment terms,as well as enrichment pathways.We also conducted microscopic experiments aiming to find key stages and reasons of unusual reproductive traits of Z pine by exploring the process of development of male and female gametes.The results are as follows:1.The embryo development of Z pine×P.massoniana has a generally resemble with that of other species of Pinus;a huge number of female gametophyte becomes atrophic in the period of April and May of the following year could mainly cause embryo abortion of the Z pine,while no pollen entering nucellus,no pollen tube growing or pollen tube discontinues growing were thought to be the secondary reasons.2.We isolated RNA from cones of P.massoniana and the Z pine of their seven stages,respectively.Illumina Hiseq X Ten was conducted for RNA sequencing.Qualified clean reads were obtained and then de novo assembled to contigs,which have a length range from 744 nt to905 nt,the range of N50 is from 1,400 nt to 1,893 nt.A total of 93,291 all-unigenes were harvested,they have an average length of 1,987 nt,the N50 is 2,494 nt.The unigenes then annotated against non-redundant nucleotide and protein databases of NCBI,Swiss-Prot,Clusters of Orthologous Groups,Gene Ontology and KEGG Orthology databases.A total of 86,006 out of 93,291 unigenes were annotated(92.19%).Unigenes of25,150(26.96%)were annotated in five databases simultaneously.We selected up LOC,SDH,ACT,EF,TOC75,DMWD,FBOX,PGK1,UBQ and CL2417C7 as candidate reference genes.We tested them across multiple tissues such as cone,young and mature stems,and young needles in both P.massoniana and Z pine.According to the evaluation results by conducting ge Norm,Norm Finder and Best Keeper,recommended and not recommended reference genes were listed for seven sample lines,respectively.Furthermore,ACT was assigned as formal reference gene for further q RT-PCR experiment of cones of P.massoniana and Z pine.3.Transcriptomic information of successive stages of cones of P.massoniana shows that differences exist among expression of stages.The number of DEGs increased in the first and the second year,respectively.The amount and increase in the second year were larger than that in the first year.MA-vs-MG has the largest number of DEGs.Significant enrichment GO terms and KEGG pathways increase gradually at later stages,which is different from the change mode of DEGs.4.Transcriptomic information of successive stages of cones of the Z pine shows that differences exist among expression of stages.The number of DEGs increase in the first and the second year,respectively.MA-vs-MG has the largest number of DEGs.The situation mentioned above is similar with that of in P.massoniana.Stage E and F could be the crucial time for making reproductive differences between P.massoniana and the Z pine,which match the results we found in microscopic study of this research.5.Transcriptomic information of successive stages of cones of P.massoniana and the Z pine shows that twenty one DEGs exist in GO term ‘response to oxidative stress',which may be a crucial clue to explore difference between them.More and more significant enrichment pathways and DEGs emerge at later stages.Expression trend type analysis shows that they have a similar components on expression trend of unigenes,though different in number of each trend profile.We found that several of DEGs indeed relate to reproductive process such as ACA7,MPK4,QRT2,TKPR1,PI5 K,PMEs,SEC6,SEC15,SWK2,PPR,EMB,LEA,SERK and BLH1.At every growth stage,these genes are expressed at a higher level in P.massoniana than in the Z pine.These phenomena may provide an explanation to unusual reproductive process in the Z pine.WGCNA(weighted gene co-expression network analysis)results show that thirty two modules were classified.Modules of ‘blueviolet' and ‘chocolate4' may provide a way insight into understanding which molecular mechanisms are altered between P.massoniana and the Z pine that might cause changes in the reproductive process.