Morphological Structures Differences And Comparative Transcriptome Analyses Between Phyllospadix Iwatensis And Zostera Marina

Seagrass is a group of higher plants submerged in seawater,which has high marine environmental adaptation and great value of ecology and fisheries.However,65%of seagrass in the world show clear degradation trend.Therefore,it is essential in practice to explore the adaptation mechanism in marine environment for seagrass protection.As a representative group of marine higher plants,Phyllospadix iwatensis Makino and Zostera marina L.are close relatives which belong to genus of Phyllospadix Hook.and Zostera L.respectively,but with distinct habitats and reproductive organs.Making clear the mechanism of these differentiation mechanism could contribute greatly to our understanding of how higher plant adapt marine environment.In this study we performed transcriptome analyses of flower buds in different developmental stages based on comprehensive habitat and morphological study of both species,focusing on the reproductive structure differences and the involved gene expression,to understand species differentiation and speciation in marine environmentWe studied the morphological and anatomical differences in roots,rhizomes,leaves,and flowers of both species,using morphological observation and paraffin section,The results showed that morphological characteristics of both species were significantly different,but each adapted to marine environment.The rhizomes of P.iwatensis were stubby,the root end inflated with dense root hairs to enhance root fixation.In contrast,the rhizomes of Z.marina were strong and long which were suitable for growing on sandy substrate.The leaves of P.iwatensis were strip and leaf sheaths were open and sheathing.There were rich mechanical tissues inside bend of leaf sheaths to enhance the resistance to sea waves.By comparison,the leaf sheaths of Z.marina were tubular.P.iwatensis was dioecious and the sex ratio of male and female plants was biased,female flowers had pistils and degenerate stamens,while the male flowers had only stamens.On the contrary,Z.marina was monoecism and the bisexual flower was composed of pistils and stamens without bracteoles or other similar structures.Gene expression of flowers at early and middle flower stage of both species was analyzed using RNA-seq method.The transcriptome of Z.marina was analyzed by reference genome,while transcriptome of P.iwatensis was analyzed de novo.Each sample generated about 50 million clean reads and a total of 2.859 billion clean bases Two contrast groups were setted,i.e.,early stage versus middle stage of Z.marina flowers,and female versus male flower at middle stage of P.iwatensis,and comparative transcriptome analyses detected 928 and 3939 differentially expressed genes,respectively,based on | log2fold change |>1,P-value<0.05.Further GO and KEGG enichment analyses showed the up-regelated expression genes in Z.marina are related to photosynthesis,anther development,and those in P.iwatensis are related to pollen and pollen tube development.Collectively,six GO entries that differ significantly between these contrast groups were detected as flower-development related genes.Notably,we found CalS5 gene have a more than 96%sequence similarity in two species and up-regulated in both contrast groups.The founding of CalS5 were further validated using qRT-PCR method.CalS5 has been found expressed specifically and highly in pollen and anthers in some model species and being considered as the callose synthase gene for pollen development in higher plants.Taken these foundings in this and previous studies together,we speculated that CalS5 could contribute to the sex differentiation of P.iwatensis.Also,CalS5 might play a key role when plants response to marine environmental stress and was a promissing gene to study phylogenetic relationship of marine submerged higher plants and their adaptation.In sum,this study explores species differentiation of P.iwatensis and Z.marina from morphological characteristics and genetic level and shows CalS5 different expression may contribute to species differentiation and reproductive isolation of two closely related species.This study also provides molecular marker for further research on evolution of plants from land to ocean and subsequent adaptation to marine environment.Furthermore,our results can also provide theoretical support for seagrass protection and ecological restoration.