- Simple sequence repeats (SSRs) are useful for genetic diversity analysis, presumably providing key information for the study and preservation of the wild populations of the two species we are interested in.. - masuca and C. - Molecular evolutionary analysis revealed functional pathways commonly enriched in unigenes with similar evolutionary rates in the two species, as well as pathways specific to each species, implicating species-specific adaptation. - The divergence time between the two closely related species was tentatively determined to be Mya.. - This is the first report of the development of expressed sequence tag (EST)-SSR markers for Calanthe. - 1 Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai 201602, China Full list of author information is available at the end of the article. - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - These species are widely distributed across the pantropical area, and southeast Asia is one of the diversity centres of Calanthe [1]. - Two of these species are from China – the widely distributed Calanthe masuca (D. - To solve this problem, Vukosavljev et al. - sinica grown in the nursery of Shanghai Chenshan Botanical Garden were used as the materials for RNA extraction and further analysis. - The cultivation and sampling times of the two species were the same to ensure reliable comparability.. - As the two species were phylogenetic- ally closely related, nucleotide sequences instead of pep- tide sequences of the unigenes were used to run a BLAST search to improve accuracy. - sinica were calculated using the method ‘GMYN’ implemented in the toolbox KaKs_Calculator (version 2.0. - EST-SSR markers validation. - Development and evaluation of EST-SSR markers. - The primers of the selected SSR alleles were synthesized and amplified, and an M13. - The universality of the developed SSR primers in Calanthe was calculated by in silico PCR. - 82,290,826 clean reads for the two species, respectively, were obtained after filtering out low-quality reads and adaptors. - With the RBH method, 25,152 pairs of putative orthologous genes between the two species were defined (Additional file 2). - sinica, of which unigenes of the two spe- cies, respectively, returned values when searched against the NR nucleotide database. - E-values and similarity dis- tributions of unigenes annotated in the NR database can be found in Table 3. - The top three species against which BlastX hits could be identified for unigenes of the two Calanthe species were Phoenix dactylifera unigenes number, the percent- age of the hit unigenes), Vitis vinifera and Oryza sativa see Additional file 3 for matched species).. - and ‘Carbohydrate transport and me- tabolism . - ‘Nuclear structure and ‘Extracellular structures . - We then mapped our unigenes of the two species onto the annotation terms of the three categories in the GO classification system, i.e., biological processes, cellular components, and molecular function. - masuca and C.. - The results showed a high degree of Table 1 Summary of the assembled transcripts of C. - consistency in GO analysis between the two species. - The top three groups with most gene members in the cat- egory of biological process were metabolic processes . - the top three largest groups in the category of cellular components were cell . - With the transcriptome data of two closely related Calanthe species available, it is possible for us to investi- gate the conservation and divergence pattern of genes in this genus, especially in the lineage represented by C.. - [26]) to define orthologous gene groups be- tween the two species studied here, using P. - We obtained the Ka, Ks, and Ka/Ks values for 10,329 orthologous gene groups between the two species, comprising 12,508 C. - The Ka/Ks value measures the average selective pressure of the two com- pared protein-coding sequences since their divergence.. - 2 Sequence length distribution of the assembled unigenes of C. - Table 3 E-value and similarity distribution of unigenes annotated in the NR database. - The top enriched GO terms in each corresponding group were quite consistent between the two species (Additional file 4). - For example, in the SP group of genes in both species, the biological process ‘oxidation-reduc- tion process’ (GO: 0055114) stood out as the most enriched GO term, with 348 and 332 associated uni- genes in C. - and ‘phosphate ion transport’ were among the top 3. - enriched GO terms in the IP group in both species. - A systematic comparison demonstrated that, in most cases, the shared enriched GO terms between corresponding gene groups of the two species outnumbered GO terms specifically enriched in each species (Table 4), which fur- ther validated the assembly and annotation of our RNA-seq data. - divergence time between the two closely related Calanthe species. - For a more robust estimation, we de- termined the minimum Ks values for each of 10,329 orthologous gene groups and determined the mode of the distribution of these Ks values at ~ 0.0328 (Fig. - The mode of distribution of the minimum Ks values of the orthologous gene groups between P. - With the divergence time between these two epidendroid orchids estimated at ~ 43 Mya [37] and assuming that the non-synonymous substitution rates between the two epidendroid and the two Calanthe spe- cies were close, we proposed that C. - This preliminary estimation of the divergence time between C. - sinica could be im- proved in the future with more data and information available.. - The majority of the SSRs in both species were either dinucleotides (1,137 in C. - Finally primer pairs were successfully desig- nated by Msatcommander for the two species. - Validation of SSR markers. - To test the amplification quality and the polymorphisms for the preliminary analysis of the 129 SSR markers, a single sample, JX1 (C. - We found that 73 of the 75 examined loci were polymorphic (97.6%) (Fig. - The two mono- morphic loci regarding these 8 individuals could represent polymorphic sites of the subpopulations from which we collected the samples for RNA sequencing. - potentially polymorphic for other uninspected populations, the two primers were excluded from further analysis for convenience. - Nonetheless, 44 out of the 73 loci were found to have a relatively high polymorphic level (PIC≥0.5) and could thus be used in population genetic studies.. - To validate the reliability of the primers of the identi- fied polymorphic loci, 13 primers with high PIC values (PIC≥0.5) were randomly selected to amplify a large number of populations and individuals (other 72 individ- uals). - The polymorphic SSR markers screening presented above was performed by comparing the transcriptomes of individuals from subpopulations in the two Calanthe species. - For the two Calanthe species, the identified primer pairs were compared be- tween them, and both were compared against two or- chids with published genomic sequences, P. - For stringency, all hits with mismatches against the two non-Calanthe orchids were excluded. - We failed to identify any primers from the two Calanthe species for P. - To a certain extent, the length of the assembly se- quence reflected the quality of the transcriptome ob- tained from the Illumina platform [43]. - The distribution of minimal synonymous substitutions per synonymous substation sites (Ks) of the orthologous gene groups between C. - catenatum determined in the publication [37] was used as real-time scale proportional to the measure of Ks. - The GO enrichment analysis carried out for each class of genes separately gave similar results in most cases, i.e., We observed much overlap in the enriched GO terms between the corresponding gene classes of the two species. - The number of specifically enriched GO terms of each category in the ID group of genes in C. - 6 The distribution of SSR type in the C. - In the two transcriptomes generated in this study putative SSRs were predicted that dif- fered in SSR number. - After searching for SSR loci among these genes, SSR loci with differences between the two transcriptomes were manually identified.. - sequenced and included in the analysis, more hits would be identified.. - A divergence time of Mya was deter- mined between the two species according to Ks. - In the future, genetic diversity studies can be conducted in the two Calanthe species and even other closely related spe- cies in this genus. - Proportion of matched unigenes in the NR database. - The authors are grateful to Ziyi Ni and Li Shao of Shanghai Chenshan Botanical Garden for their help in caring for the materials in the greenhouse.. - We all thank Miao An for the help in the data analysis.. - Zhai JW, Zhang GQ, Li L, Wang M, Chen LJ, Chung SW, et al. - A new phylogenetic analysis sheds new light on the relationships in the Calanthe alliance (Orchidaceae) in China. - Leitch IJ, Kahandawala I, Suda J, Hanson L, Ingrouille MJ, Chase MW, et al.. - Parida SK, Kalia SK, Kaul S, Dalal V, Hemaprabha G, Selvi A, et al. - Tang J, Baldwin SJ, Jacobs JM, van der Linden CG, Voorrips RE, Leunissen JA, et al. - Castoe TA, Poole AW, de Koning AJ, Jones KL, Tomback DF, Oyler-McCance SJ, et al. - Development and validation of EST-SSR markers from the transcriptome of adzuki bean (Vigna angularis). - Jiao Y, Jia HM, Li XW, Chai ML, Jia HJ, Chen Z, et al. - Mahajan V, Rather IA, Awasthi P, Anand R, Gairola S, Meena SR, et al.. - Vukosavljev M, Esselink GD, van 't Westende WP, Cox P, Visser RG, Arens P, et al. - Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. - Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al.. - Xie C, Mao X, Huang J, Ding Y, Wu J, Dong S, et al. - Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al.. - Cai J, Liu X, Vanneste K, Proost S, Tsai WC, Liu KW, et al. - The genome sequence of the orchid Phalaenopsis equestris. - Zhang GQ, Xu Q, Bian C, Tsai WC, Yeh CM, Liu KW, et al. - Chehab EW, Kaspi R, Savchenko T, Rowe H, Negre-Zakharov F, Kliebenstein D, et al. - Zhang GQ, Liu KW, Li Z, Lohaus R, Hsiao YY, Niu SC, et al. - Wei W, Qi X, Wang L, Zhang Y, Hua W, Li D, et al. - Characterization of the sesame (Sesamum indicum L.) global transcriptome using Illumina paired- end sequencing and development of EST-SSR markers. - Zhang J, Wu K, Zeng S, Teixeira da Silva JA, Zhao X, Tian CE, et al.. - Su C, Chao Y, Chang A, Chen W, Chen C, Lee A, et al. - De novo assembly of expressed transcripts and global analysis of the Phalaenopsis aphrodite transcriptome. - Zhou X, Dong Y, Zhao J, Huang L, Ren X, Chen Y, et al
Xem thử không khả dụng, vui lòng xem tại trang nguồn hoặc xem
Tóm tắt