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Utility of PacBio Iso-Seq for transcript and gene discovery in Hevea latex

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Abstract

Full-length cDNA libraries (< 1 kb, 1–2 kb, 2–3 kb, 3–6 kb size ranges) were generated from latex of a Hevea brasiliensis tree clone, RRIM 600, for PacBio Iso-Seq sequencing. The total number of high-quality consensus sequences obtained from nearly 20 Gb of raw reads was 207,943 having an average length of 1823 bp and N50 of 2538 bp. Mapping of high-quality consensus sequences to the published Reyan 7-33-97 genome sequence produced 37,224 Iso-Seq transcripts with an average length of 2030 bp and N50 of 2529 bp. These represented 14,999 genic loci. Comparison with the Reyan 7-33-97 gene models indicated that 50.75% of Iso-Seq transcripts were potentially novel gene isoforms. Subsequently, we also demonstrate the application of Iso-Seq in identifying previously unknown alternatively spliced transcripts based on three case studies of candidate rubber genes of interest. Findings from this study support the usefulness of PacBio Iso-Seq as transcriptome resources for gene family characterisation and genome annotation in Hevea.

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References

  1. An D, Cao H, Li C, Humbeck K, Wang W (2018) Isoform sequencing and state-of-art applications for unravelling complexity of plant transcriptomes. Genes 9(1):43

    Google Scholar 

  2. Berthelot K, Lecomte S, Estevez Y, Peruch F (2014) Hevea brasiliensis REF (Hev b 1) and SRPP (Hev b 3): an overview on rubber particle proteins. Biochimie 106:1–9

    CAS  Google Scholar 

  3. Bokma E, Barends T, Terwissch van Scheltingab AC, Dijkstr BW, Beintema JJ (2000) Enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis. FEBS Lett 478:119–122

    CAS  Google Scholar 

  4. Bokma E, Rozeboom HJ, Sibbald M, Dijkstra BW, Beintema JJ (2002) Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis. Eur J Biochem 269:893–901

    CAS  Google Scholar 

  5. Bokma E, Spiering M, Chow KS, Mulder PP, Subroto T, Beintema JJ (2001) Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis. Plant Physiol Biochem 39:367–376

    CAS  Google Scholar 

  6. Bokma E, van Koningsveld GA, Jeronimus-Stratingh M, Beintema JJ (1997) Hevamine, a chitinase from the rubber tree Hevea brasiliensis, cleaves peptidoglycan between the C-1 of N-acetylglucosamine and C-4 of N-acetylmuramic acid and therefore is not a lysozyme. FEBS Lett 411(2–3):161–163

    CAS  Google Scholar 

  7. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+ : architecture and applications. BMC Bioinform 10:421

    Google Scholar 

  8. Chao J, Chen Y, Wu S, Tian WM (2015) Comparative transcriptome analysis of latex from rubber tree clone CATAS8-79 and PR107 reveals new cues for the regulation of latex regeneration and duration of latex flow. BMC Plant Biol 15(1):104

    Google Scholar 

  9. Cheng B, Furtado A, Henry RJ (2017) Long-read sequencing of the coffee bean transcriptome reveals the diversity of full-length transcripts. Gigascience 6(11):gix86

    Google Scholar 

  10. Chow KS, Wan KL, Mat Isa MN, Bahari A, Tan SH, Harikrishna K, Yeang HY (2007) Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex. J Exp Bot 58:2429–2440

    CAS  Google Scholar 

  11. Chow KS, Mat Isa MN, Bahari A, Ghazali AK, Alias H, Mohd-Zainuddin Z, Hoh CC, Wan KL (2012) Metabolic routes affecting rubber biosynthesis in Hevea brasiliensis latex. J Exp Bot 63:1863–1871

    CAS  Google Scholar 

  12. Chow KS, Ghazali AK, Hoh CC, Mohd-Zainuddin Z (2014) RNA sequencing read depth requirement for optimal transcriptome coverage in Hevea brasiliensis. BMC Res Notes 7:69

    Google Scholar 

  13. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676

    CAS  Google Scholar 

  14. Dong L, Liu H, Zhang J, Yang S, Kong G, Chu JSC, Chen N, Wang D (2015) Single-molecule real-time transcript sequencing facilitates common wheat genome annotation and grain transcriptome research. BMC Genom 16:1039

    Google Scholar 

  15. Duan C, Argout X, Gébelin V, Summo M, Dufayard JF, Leclercq J, Piyatrakul P, Pirrello J, Rio M, Champion A, Montoro P (2013) Identification of the Hevea brasiliensis AP2/ERF superfamily by RNA sequencing. BMC Genom 14(1):30

    CAS  Google Scholar 

  16. Filichkin SA, Hamilton M, Dharmawardhana PD, Singh SK, Sullivan C, Ben-Hur A, Reddy AS, Jaiswal P (2018) Abiotic stresses modulate landscape of poplar transcriptome via alternative splicing, differential intron retention, and isoform ratio switching. Front Plant Sci 12(9):5

    Google Scholar 

  17. Gordon SP, Tseng E, Salamov A, Zhang J, Meng X, Zhao Z, Kang D, Underwood J, Grigoriev IV, Figueroa M, Schilling JS, Chen F, Wang Z (2015) Widespread polycistronic transcripts in fungi revealed by single-molecule mRNA sequencing. PLoS One 10:e0132628

    Google Scholar 

  18. Goyvaerts E, Dennis M, Light D, Chua NH (1991) Cloning and sequencing of the cDNA encoding the rubber elongation factor of Hevea brasiliensis. Plant Physiol 97(1):317–321

    CAS  Google Scholar 

  19. Hoang NV, Furtado A, Mason PJ, Marquardt A, Kasirajan L, Thirugnanasambandam PP, Botha FC, Henry RJ (2017) A survey of the complex transcriptome from the highly polyploid sugarcane genome using full-length isoform sequencing and de novo assembly from short read sequencing. BMC Genom 18(1):395

    Google Scholar 

  20. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17(8):754–755

    CAS  Google Scholar 

  21. Jekel PA, Hartmann BH, Beintema JJ (1991) The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex. Eur J Biochem 200:123–130

    CAS  Google Scholar 

  22. Jiao WB, Schneeberger K (2017) The impact of third generation genomic technologies on plant genome assembly. Curr Opin Plant Biol 36:64–70

    CAS  Google Scholar 

  23. Jo IH, Lee J, Hong C, Lee D, Bae W, Park SG, Ahn Y, Kim Y, Kim J, Lee J, Hyun D (2017) Isoform sequencing provides a more comprehensive view of the panax ginseng transcriptome. Genes 8(9):228

    Google Scholar 

  24. Ko JH, Chow KS, Han KH (2003) Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers of Hevea brasiliensis (para rubber tree). Plant Mol Biol 53:479–492

    CAS  Google Scholar 

  25. Lau NS, Makita Y, Kawashima M, Taylor TD, Kondo S, Othman AS, Chong ASC, Matsui M (2016) The rubber tree genome shows expansion of gene family associated with rubber biosynthesis. Sci Rep 6:28594

    CAS  Google Scholar 

  26. Li C, Lin F, An D, Wang W, Huang R (2017) Genome sequencing and assembly by long reads in plants. Genes 9(1):6

    Google Scholar 

  27. Li Y, Wei W, Feng J, Luo H, Pi M, Liu Z, Kang C (2017) Genome re-annotation of the wild strawberry Fragaria vesca using extensive Illumina-and SMRT-based RNA-seq datasets. DNA Res 25(1):61–70

    Google Scholar 

  28. Liu JP, Xia ZQ, Tian XY, Li YJ (2015) Transcriptome sequencing and analysis of rubber tree (Hevea brasiliensis Muell.) to discover putative genes associated with tapping panel dryness (TPD). BMC Genom 16(1):398

    Google Scholar 

  29. Lopez D, Amira MB, Brown D, Muries B, Brunel-Michac N, Bourgerie S, Porcheron B, Lemoine R, Chrestin H, Mollison E, Di Cola A, Frigerio L, Julien JL, Gousset-Dupont A, Fumanal B, Label P, Pujade-Renaud V, Auguin D, Venisse JS (2016) The Hevea brasiliensis XIP aquaporin subfamily: genomic, structural and functional characterizations with relevance to intensive latex harvesting. Plant Mol Biol 91:375–396

    CAS  Google Scholar 

  30. Mantello CC, Cardoso-Silva CB, Da Silva CC, De Souza LM, Scaloppi EJ Jr, de Goncalves PS, Vicentini R, De Souza AP (2014) De novo assembly and transcriptome analysis of the rubber tree (Hevea brasiliensis) and SNP markers development for rubber biosynthesis pathways. PLoS One 9:e102664

    Google Scholar 

  31. Montoro P, Wu S, Favreau B, Herlinawati E, Labrune C, Martin-Magniette ML, Pointet S, Rio M, Leclercq J, Ismawanto S (2018) Transcriptome analysis in Hevea brasiliensis latex revealed changes in hormone signalling pathways during ethephon stimulation and consequent Tapping Panel Dryness. Sci Rep 8(1):8483

    Google Scholar 

  32. Oh SK, Kang H, Shin DH, Yang J, Chow KS, Yeang HY, Wagner B, Breiteneder H, Han KH (1999) Isolation, characterization, and functional analysis of a novel cDNA clone encoding a small rubber particle protein from Hevea brasiliensis. J Biol Chem 274(24):17132–17138

    CAS  Google Scholar 

  33. Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL (2016) Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Protoc 11:1650–1667

    CAS  Google Scholar 

  34. Pootakham W, Sonthirod C, Naktang C, Ruang-Areerate P, Yoocha T, Sangsrakru D, Theerawattanasuk K, Rattanawong R, Lekawipat N, Tangphatsornruang S (2017) De novo hybrid assembly of the rubber tree genome reveals evidence of paleotetraploidy in Hevea species. Sci Rep 7:41457

    CAS  Google Scholar 

  35. Roberts RJ, Carneiro MO, Schatz MC (2013) The advantages of SMRT sequencing. Genome Biol 14(6):405

    Google Scholar 

  36. Roberts A, Trapnell C, Donaghey J, Rinn JL, Pachter L (2011) Improving RNA-Seq expression estimates by correcting for fragment bias. Genome Biol 12:R22

    CAS  Google Scholar 

  37. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29(1):24

    CAS  Google Scholar 

  38. Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61(3):539–542

    Google Scholar 

  39. Rozeboom HJ, Budiani A, Beintema JJ, Dijkstra BW (1990) Crystallization of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex. J Mol Biol 212:441–443

    CAS  Google Scholar 

  40. Salgado LR, Koop DM, Pinheiro DG, Rivallan R, Le Guen V, Nicolás MF, De Almeida LG, Rocha VR, Magalhães M, Gerber AL, Figueira A (2014) De novo transcriptome analysis of Hevea brasiliensis tissues by RNA-seq and screening for molecular markers. BMC Genom 15(1):236

    Google Scholar 

  41. Schulz MH, Zerbino DR, Vingron M, Birney E (2012) Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels. Bioinformatics 28:1086–1092

    CAS  Google Scholar 

  42. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7(1):539

    Google Scholar 

  43. Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM (2015) BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31:3210–3212

    Google Scholar 

  44. Subroto T, van Koningsveld GA, Schreuder HA, Soedjanaatmadja UM, Beintema JJ (1996) Chitinase and β-1, 3-glucanase in the lutoid-body fraction of Hevea latex. Phytochemistry 43(1):29–37

    CAS  Google Scholar 

  45. Tan D, Hu X, Fu L, Kumpeangkeaw A, Ding Z, Sun X, Zhang J (2017) Comparative morphology and transcriptome analysis reveals distinct functions of the primary and secondary laticifer cells in the rubber tree. Sci Rep 7(1):3126

    Google Scholar 

  46. Tang C, Xiao X, Li H, Fan Y, Yang J, Qi J, Li H (2013) Comparative analysis of latex transcriptome reveals putative molecular mechanisms underlying superproductivity of Hevea brasiliensis. PLoS One 8:e75307

    CAS  Google Scholar 

  47. Tang C, Yang M, Fang Y, Luo Y, Gao S, Xiao X, An Z, Zhou B, Zhang B, Tan X, Yeang HY, Qin Y, Yang J, Lin Q, Mei H, Montoro P, Long X, Qi J, Hua Y, He Z, Sun M, Li W, Zeng X, Cheng H, Liu Y, Yang J, Tian W, Zhuang N, Zeng R, Li D, He P, Li Z, Zou Z, Li S, Li C, Wang J, Wei D, Lai CQ, Luo W, Yu J, Hu S, Huang H (2016) The rubber tree genome reveals new insights into rubber production and species adaptation. Nat Plants 2:16073

    CAS  Google Scholar 

  48. Terwisscha van Scheltinga AC, Hennig M, Dijkstra BW (1996) The 1.8 A resolution structure of hevamine, a plant chitinase/lysozyme, and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18. J Mol Biol 262:243–257

    CAS  Google Scholar 

  49. Terwisscha van Scheltinga AC, Kalk KH, Beintema JJ, Dijkstra BW (1994) Crystal structures of hevamine, a plant defence protein with chitinase and lysozyme activity, and its complex with an inhibitor. Structure 2:1181–1189

    CAS  Google Scholar 

  50. Thorvaldsdóttir H, Robinson JT, Mesirov JP (2013) Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14(2):178–192

    Google Scholar 

  51. Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L (2013) Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol 31:46–53

    CAS  Google Scholar 

  52. Tungngoen K, Viboonjun U, Kongsawadworakul P, Katsuhara M, Julien JL, Sakr S, Chrestin H, Narangajavana J (2011) Hormonal treatment of the bark of rubber trees (Hevea brasiliensis) increases latex yield through latex dilution in relation with the differential expression of two aquaporin genes. J Plant Physiol 168(3):253–262

    CAS  Google Scholar 

  53. Wang B, Tseng E, Regulski M, Clark TA, Hon T, Jiao Y, Lu Z, Olson A, Stein JC, Ware D (2016) Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing. Nat Commun 7:11708

    CAS  Google Scholar 

  54. Wang M, Wang P, Liang F, Ye Z, Li J, Shen C, Pei L, Wang F, Hu J, Tu L, Lindsey K (2018) A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation. New Phytol 217(1):163–178

    Google Scholar 

  55. Wu TD, Watanabe CK (2005) GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21:1859–1875

    CAS  Google Scholar 

  56. Xia Z, Xu H, Zhai J, Li D, Luo H, He C, Huang X (2011) RNA-Seq analysis and de novo transcriptome assembly of Hevea brasiliensis. Plant Mol Biol 77:299–308

    CAS  Google Scholar 

  57. Yuan Y, Bayer PE, Batley J, Edwards D (2017) Improvements in genomic technologies: application to crop genomics. Trends Biotechnol 35(6):547–558

    CAS  Google Scholar 

  58. Zerbino DR (2010) Using the velvet de novo assembler for short-read sequencing technologies. Curr Protoc Bioinform 31(1):11.5.1–11.5.12

    Google Scholar 

  59. Zhang W, Ciclitira P, Messing J (2013) PacBio sequencing of gene families—a case study with wheat gluten genes. Gene 533:541–546

    Google Scholar 

  60. Zhang G, Sun M, Wang J, Lei M, Li C, Zhao D, Huang J, Li W, Li S, Li J, Yang J (2019) PacBio full-length cDNA sequencing integrated with RNA-seq reads drastically improves the discovery of splicing transcripts in rice. Plant J 97(2):296–305

    CAS  Google Scholar 

  61. Zhu C, Li X, Zheng J (2018) Transcriptome profiling using Illumina-and SMRT-based RNA-seq of hot pepper for in-depth understanding of genes involved in CMV infection. Gene 666:123–133

    CAS  Google Scholar 

  62. Zou Z, Gong J, An F, Xie G, Wang J, Mo Y, Yang L (2015) Genome-wide identification of rubber tree (Hevea brasiliensis Muell. Arg.) aquaporin genes and their response to ethephon stimulation in the laticifer, a rubber-producing tissue. BMC Genom 16:1001

    Google Scholar 

  63. Zuo C, Blow M, Sreedasyam A, Kuo RC, Ramamoorthy GK, Torres-Jerez I, Li G, Wang M, Dilworth D, Barry K, Udvardi M (2018) Revealing the transcriptomic complexity of switchgrass by PacBio long-read sequencing. Biotechnol Biofuels 11(1):170

    Google Scholar 

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Acknowledgements

The authors wish to thank the Malaysian Rubber Board for financial support and Mr. V. Monyrajan for excellent field assistance during sample collection.

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Authors and Affiliations

  1. Production Development Division, Malaysian Rubber Board, Experiment Station, 47000, Sungai Buloh, Selangor, Malaysia

    K.-S. Chow & Z. Mohd.-Zainuddin

  2. Codon Genomics, 7, Jalan Dutamas, Balakong, 43200, Cheras, Selangor, Malaysia

    J.-S. Khoo, S.-M. Ng & C.-C. Hoh

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  1. K.-S. Chow
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Contributions

K-SC, J-SK and C-CH conceptualized the study. K-SC, J-SK are co-first authors of this paper. ZM-Z co-ordinated latex collections and performed latex RNA isolations. J-SK and S-MN executed all bioinformatics analysis. J-SK and KS-C wrote the manuscript with input from all authors.

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Correspondence to K.-S. Chow.

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Chow, KS., Khoo, JS., Mohd.-Zainuddin, Z. et al. Utility of PacBio Iso-Seq for transcript and gene discovery in Hevea latex. J Rubber Res 22, 169–186 (2019). https://doi.org/10.1007/s42464-019-00026-7

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