| The ferns originally evolved as a widely distributed and ancient vascular plant with a distinctive life cycle in which the haploid and diploid generations are completely separated, representing a critical clade in land plants.The ferns are widely used in medicine, food, industry and landscaping. At present, the studies are mainly focused on plant taxonomy, phylogeny, resources collection and evalution and genetic diversity analysis and so on, but DNA C-value estimation, genome datas, transcriptome sequencing and analysis are scarce, resulting in the lagging of developing molecular markers, cloning genes and molecular phylogenetic research. The paper choose Osmunda japonica belonging to Protoleptosporangiopsida and Hypolepis punctate belonging to Leptosporangiopsida:on the basis of optimizing the FCM estimation methods of C value, we estimated C values of O.japonica and H.punctate and we choose the sporophyte leaves of O.japonica and H.punctate as the experimental material to conduct transcriptome sequencing and analysising through the Illumina high-through-put sequencing technology. The transcriptome databases of O.japonica and H.Punctate were firstly established. The main results were as below:1. We used fresh and silica gel-dried leaves of O japonica as materials, Otto and GPB as extraction buffers to optimize and explore the estimation methods of DNA C value for O.japonica using FCM, and on this basis we estimated the DNA C value of H.Punctate.The results showed:the optimal condition is determined as that using Oryza sativa as internal standard and silica gel-dried leaves as materials, and using Otto’s buffer with two-steps method to extract, prepare and store under low temperature with the concentration of propidium iodide (PI) at 20 ug/mL. The estimation results according to above procedure showed better results were easily and accurately gotten with relatively concentrated scatterplot, less debris, more simplicity to take gates and lower coefficient of variance (CV). Finally, the C value of O.japonica was first estimated successfully as 2C=17.15pg by FCM. Based on the optimal estimation system of O.japonica,we select fresh leaves of H.punctate and use Oryza sativa as internal standard as materials and the estimation conditions were same as the O.Japonica’s. According to above procedure, the estimation results showed better results were easily and accurately gotten with relatively concentrated scatterplot, less debris, more simplicity to take gates and lower coefficient of variance (CV). The C value of H.Punctate was first estimated successfully as 2C=5.98pg by FCM.2. After the extraction of the sample RNA, preparation of the cDNA library, Illumina sequencing, filtration of the failure sequence, and de novo assembly using Trinity, eventually, we got 44348 of O.japonica Unigenes, which an average length is 864bp and N50 length is 1687bp. In the same way, we got 44542 of H.punctate Unigenes, which an average length is 715bp and N50 length is 1216bp. The test showed that assembly quality and length of sequencing datas meet the basic requirements of transcriptome analysis.3. To identify the putative functions of the Unigenes, we performed Blast alignmen-ts aganist the with the public databases (NT, NR, KO, swissprot, Pfam, GO, K.OG), there were 21225 unigenes(47.86% of all) with matches to one or more of the database for O.Japonica and there were 24285 unigenes(54.52% of all)with matches to one or more of the database for H.punctate. Comparing with the COG database, O.japonica and H.punctate were both divided into 26 different gene functional annotation, which the O.japonica genes mostly involved in general function prediction only(1457), Post-translational modification、protein turnover、chaperon (1028), Signal Transducti-on(693) and the H.punctate genes also mostly involved in general function prediction only(1522), Post-translational modification、protein turnover、chaperon (1116), Signal Transduction(849).The distribution of the annotated unigenes in each functional group was alike for O.japonica and H.punctate. Through GO and Pathway enrichment analysis, the O.japonica and the H.punctate Unigenes were respectively made to get 48 and 47 different functional categories, which involved in 242 and 244 metabolic pathways, respectively. We made a CDS forecast by the Blast and by ESTScan software for O.japonica and H.punctate. The O japonica got a total of 42503 CDS; The H. punctate got a total of 42756 CDS. We also counted the length distribution of the CDS obtained by the Blast and ESTScan, and we find that the length of CDS obtained by ESTScan are less than the length of CDS obtained by Blast. The establishment of O. japonica and H. punctate transcriptome dababases will lay foundation for further gene combination and functional genomics research.4. Through the SSR analysis of O.japonica and H.punctate, O.japonica get a total of 9639 SSR markers among 7470 Unigenes. The mononucleotide repeats, diriucleotide repeats and trinucleotide repeats which SSR types are majorly distributed account for 96.72% of the total markers, the left quadnucleotide repeats, pentanucleotide repeats, hexanucleotide repeats account for 3.27%. H.punctate get a total of 8626 SSR markers among 6803 Unigenes. The diriucleotide repeats which SSR types are majorly distributed account for 75.74% of the total markers, the left mononucleotide repeats, quadnucleotide repeats, trinucleotide repeats, pentanucleotide repeats, hexanucleotide repeats account for 24.26%. SSR analysis will lay the foundation for the follow-up studies such as the analysis of the genetic diversity, construction of genetic linkage map and molecular breeding and so on.5. Through the flavonoid synthesis pathway analysis of O.japonica and H. punctate, the obtained O.japonica Unigenes had 182,92 annotated in phenylpropanoid biosynthesis, flavonoid biosynthesis, respectively, which involves in 26 kinds of ungenes. H.punctate Unigenes had 108,48 annotated in phenylpropanoid biosynthesis, flavonoid biosynthesis, respectively, which involves in 22 kinds of ungenes.There are8, 5,15,7,1 annotated in Phenylalanine ammonia-lyase,4-Coumarate-CoA-ligase, Trans-cinnamate4-monooxygenase, Chalcone synthase, Chalcone isomerase for O.japonica. There are 11,5,9,6,1 annotated in PAL,4CL, CYP73A, CHS and CHI for H.punctate. Exploring the flavonoids synthesis related genes will lay the important foundation for research on the key gene cloning and the secondary metabolites biosynthesis pathway etc. |
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