Genetic And Non-Genetic Divergence Of Parrotia Subaequalis Seedlings From Isolated Provenances Grown In A Common Garden

Parrotia subaequalis is a unique deciduous tree of the genus Hamamelidaceae in China.It is considered as a national Grade I key protected plant due to its exceptional taxonomic status and narrow distribution in wild.It is also recognized by the International Union for Conservation of Nature(IUCN)as an extremely endangered species.There are many facts leading to the survival crisis of Parrotia subaequalis,such as climate change,artificial felling,habitat loss and the like;internal factors include pollination difficulties,low seed setting rate and low photosynthetic competitiveness.As a key species in the phylogenetic research of the genus Hamamelidaceae,previous research focused on its field survival status,population structure and spatial distribution,interspecific relationships,in-field breeding,ex situ conservation and greening applications,artificial breeding and propagation,nutrient analysis,genetic and phenotypic differentiation.Concerning this,a comprehensive research on Parrotia subaequalis concerning phynotypic traits,physiological and photosynthetic functions and autumn leaf coloring mechanisms is to be performed.In our experiment,seeds of Parrotia subaequalis collected from seven natural distribution spots are cultivated in one common garden with a mild climate and in proper environment.Leaves of the seedlings were used as research materials for measurement of phenotypic traits,physiological and photosynthesis parameters as well as pigment concentration,in order to reveal inheritable phenotypic,physiological and fuctional variation of the individuals and to quantify diverse leaf color transformation in autumn.Transcriptome sequencing(RNA Seq)was further applied to anchor the pigment-or photosynthesis-related genes,and to discuss about the expression levels of these genes.As a result,key genes controlling the formation of the leaf color of Parrotia subaequalis were screened and measured.The crucial findings are as follows:1.Seed germination monitoring,plant biomass measurement and leaf element analysis showed that the germination rate,the survival rate and the content of leaf carbon,nitrogen and hydrogen of individuals from the Yanzi River Grand Canyon in Jinzhai,Anhui Province(AJY)rank first,while the germination energy(26%,1%lower than the highest)ranked second,indicating that the seed activity and germination ability of this provenance are the greatest,and the absorption rate,assimilation rate of soil elements and carbon dioxide also mark the highest;the germination and survival rate of the other 6 groups were close,indicating that the germination status of the seeds was synchronized and the seedling emergence was consistent.During the monitoring period,the gross number of seedling death of the 7 groups accounted for only 2.17%of the gross number of seeds,fitting in a low level of death rate.These statistics also demonstrated the important premise that the seeds were qualified and the environment of the common garden was suitable for the plants to grow.2.Quantification of leaf phenotypic traits showed that the leaf index(LI)of the7 groups could be significantly(p<0.05)divided into two categories,corresponding to the elongated shape(LI=2.0)and oval(LI=1.75)leaves;leaf area(LA)and specific leaf area(SLA)of leaves from AYT plant in Toutuo,Yuexi,Anhui Province were the highest,while leaf thickness(LT),leaf dry matter content(LDMC)of this site are the lowest,indicating these leaves being thin and large,and the leaf tissue being more hydrated,and the dry matter content being lower.The leaves of Forest Farm,Yixing,Jiangsu(JYL)are the thickest(0.271±0.0025mm)and their leaf area rank the second largest(11.313±0.7402cm2),indicating these leaves being thick and large;in addition,the AJY individu als of the highest seed germination rate and the second highest germination energy present an appropriate level of leaf morphological indexes.3.The fitting results of light response curves show that the light compensation points of the 7 groups are in the range of 34-78 ?mol·m-2·s-1,with the light saturation point in the range of 320-500?umol·m-2·s-1;the respiration rate in the range of 1.6-3.5 mg(?l)·h-1 g-1).The fitting curves show that the maximum net photosynthetic rate of leaves in Forest Farm,Yixing,Jiangsu(JYL)is the highest,while that of the Yanzi River Grand Canyon,Jinzhai,Anhui(AJY)is the lowest,and that of Shanjuandong,Yixing,Jiangsu(JYS)individuals rank the second lowest,indicating that individuals originating from Forest Farm,Yixing,Jiangsu(JYL)have the highest light utilization rate,which is significantly higher than other groups.The photosynthesis parameters and chlorophyll fluorescence parameters indicated that the chlorophyll content of Longwangshan,Anji,Zhejiang(ZAL),Huangwei,Yuexi,Anhui(AYH)and Forest Farm,Yixing,Jiangsu(JYL)have great ability to utilize medium sunlight.4.During the leaf color turning period in autumn,leaves of the two provenances,Yanzi River Grand Canyon,Jinzhai,Anhui(AJY)and Toutuo,Yuexi,Anhui(AYT),turned red(redness:aAJY=53.253±1.5289;aAlT=52.96±1.2332);leaves of Huangwei,Yuexi,Anhui(AYH)turned pink(aAYH=12.543±0.0339);while leaves of Forest Farm,Yixing,Jiangsu(JYL)and Shanjuandong,Yixing,Jiangsu(JYS)turned yellow(yellowness:bJYL=52.303±1.2953;bJYS=51.643±1.6933);leaves of Zhuxian,Jixi,Anhui(AJZ)and Longwangshan,Anji,Zhejiang(ZAL)turned purple(LAJZ=29.323±0.1366 bAJZ=3.028±0.1816;LZAL=29.37±0.1044 bZAL=3.013±0.2021).The measurement of pigment contents showed that anthocyanins in leaves are related to the color of purple(?2.4mg/g),red(2mg/g)and pink(1.4mg/g).5.Net photosynthetic rate(Parln=700?mol·m-2·s-1)measured in the autumn leaf coloring stage showed that the photosynthetic capacity of purple leaves(PnAJZ=8.498±1.2343;PnzAL=8.384±0.5112)was significantly stronger than that of red(PnAjY=7.891).±1.0525;PnAYT=6.156±0.3216)and pink(PnAYH=7.021±0.492)leaves,while the photosynthetic capacity of red and pink leaves being significantly stronger than that of yellow leaves(PnjYL=4.506±0.8018;PnJYS=5.312±0.2371).6.RNA-seq technology was applied to sequence and analyze the transcriptome of four-color leaves of Parrotia subaequalis,basing on the Illumina second-generation high-throughput sequencing platform with PE150 sequencing strategy.As a result,a total of 109112387 raw read pairs were screened out.After quality inspection and quality control screening,106592998 clean read pairs were obtained,and the calibration rate was 97.7%The clean read pairs were compared between every two group.After FPKM converting,7533 differentially expressed genes were eventually identified.Basing on several databases,we aimed to anchor functional genes with direct or indirect relationship to leaf photosynthesis and coloration.Some unigenes with different expression levels in different groups are relevant to plant functions involving photosystem I assembly coding protein Ycf3(c328128),photosystem II D2 protein(GeneiD:c327135),blue light photoreceptor(GeneID:c324989),photosynthetic electron transport(GeneI D:c304440,c327135),thylakoid formation(GeneID:c297542),chloroplast protein,photosystem II assembly(GeneID:c297542)and the like.The results of DEGs were further analyzed with GO and KEGG database,implying that the purple and red leaves are in an active state at the transcriptional level,while the yellow leaves may have entered the apoptotic stage.The transcriptional activity of red and pink leaves was lower than that of purple leaves and higher than that of yellow leaves,but with no significant difference between them.