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Molecular spectrum of somaclonal variation in PLB-regenerated Oncidium revealed by SLAF-seq

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Abstract

Protocorm-like body (PLB) formation is an efficient method that has been widely used in the propagation of orchids. There is little information regarding the genome-wide molecular spectrum of somaclonal variation in PLB-regenerated orchids. We used specific-locus amplified fragment sequencing (SLAF-seq) to investigate the variations of single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) in somaclones of Oncidium ‘Milliongolds’, which was regenerated via PLB formation after in vitro culturing for 10 years. A high average variation rate (1.00 × 10− 3) was detected in 11 somaclones, and the most abundant variations were line specific. The average Ti/Tv ratio was 2.17, and G:C > C:G was the rarest type of SNP. The SNPs adjacent to adenine and thymine were more frequent than those adjacent to guanine and cytosine. The most prominent InDels were mononucleotide InDels. More than half of the InDels occurred within or adjacent to homopolymeric or polynucleotide repeats, and a majority of the repeats were thymine or adenine homopolymers. This study expands the current understanding of the somaclonal variation that occurs in PLB regeneration systems and long-term subcultures, which will be useful for improving the use of PLB formation in the propagation and breeding of orchids. We also confirmed that SLAF-seq is an efficient technology for genome-wide analysis of somaclonal variations for most species without reference genome sequences.

Key message

We expanded the understanding of somaclonal variation in PLB regeneration and long-term subcultures of orchids, and confirmed SLAF-seq is efficient for genome-wide analysis of somaclonal variations without reference genome sequences.

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Data availability

The Illumina SLAF-seq data developed from Oncidium are available at the NCBI Sequence Read Archive database with study accession number SRP159117.

Abbreviations

PLB:

Protocorm-like body

RFLP:

Restriction fragment length polymorphism

RAPD:

Randomly amplified polymorphic DNA

DNA:

Deoxyribonucleic acid

AFLP:

Amplified fragment length polymorphism

ISSR:

Inter simple sequence repeat

SSR:

Simple sequence repeat

NGS:

Next-generation sequencing

SNP:

Single nucleotide polymorphism

InDel:

Insertion-deletion

SLAF-seq:

Specific-locus amplified fragment sequencing

RRL:

Reduced representation library

GC:

Guanine-cytosine

Ti:

Transition

Tv:

Transversion

A:

Adenine

T:

Thymine

G:

Guanine

C:

Cytosine

cDNA:

Complementary deoxyribonucleic acid

PCR:

Polymerase chain reaction

MS:

Murashige–Skoog

BA:

N6-benzyladenine

NAA:

α-Naphthalene acetic acid

References

  • Agarwal T, Gupta AK, Patel AK, Shekhawat NS (2015) Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers. Plant Cell Tissue Organ Cult 120:313–323

    Article  CAS  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  • Bairu MW, Aremu AO, Staden JV (2011) Somaclonal variation in plants: causes and detection methods. Plant Growth Regul 63:147–173

    Article  CAS  Google Scholar 

  • Barret P, Brinkman M, Beckert M (2006) A sequence related to rice Pong transposable element displays transcriptional activation by in vitro culture and reveals somaclonal variations in maize. Genome 49:1399–1407

    Article  CAS  PubMed  Google Scholar 

  • Belfield EJ, Gan X, Mithani A, Brown C, Jiang C, Franklin K, Alvey E, Wibowo A, Jung M, Bailey K, Kalwani S, Ragoussis J, Mott R, Harberd NP (2012) Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana. Genome Res 22:1306–1315

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cai J, Liu X, Vanneste K, Proost S, Tsai WC, Liu KW, Chen LJ, He Y, Xu Q, Bian C, Zheng Z, Sun F, Liu W, Hsiao YY, Pan ZJ, Hsu CC, Yang YP, Hsu YC, Chuang YC, Dievart A, Dufayard JF, Xu X, Wang JY, Wang J, Xiao XJ, Zhao XM, Du R, Zhang GQ, Wang M, Su YY, Xie GC, Liu GH, Li LQ, Huang LQ, Luo YB, Chen HH, Van de Peer Y, Liu ZJ (2015) The genome sequence of the orchid Phalaenopsis equestris. Nat Genet 47:65–72

    Article  CAS  PubMed  Google Scholar 

  • Chai ML, Xu CJ, Senthil KK, Kim JY, Kim DH (2002) Stable transformation of protocorm-like bodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens. Sci Hortic 96:213–224

    Article  CAS  Google Scholar 

  • Chen JT, Chang WC (2004) TIBA affects the induction of direct somatic embryogenesis from leaf explants of Oncidium. Plant Cell Tissue Organ Cult 79:315–320

    Article  CAS  Google Scholar 

  • Chen WH, Chen TM, Fu YM, Hsieh RM, Chen WS (1998) Studies on somaclonal variation in Phalaenopsis. Plant Cell Rep 18:7–13

    Article  Google Scholar 

  • Chen YH, Tsai YJ, Huang JZ, Chen FC (2005) Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture. Cell Res 15:639–657

    Article  CAS  PubMed  Google Scholar 

  • Chin DP, Mishiba K, Mii M (2007) Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium. Plant Cell Rep 26:735–743

    Article  CAS  PubMed  Google Scholar 

  • Chugh S, Guha S, Rao IU (2009) Micropropagation of orchids: a review on the potential of different explants. Sci Hortic 122:507–520

    Article  CAS  Google Scholar 

  • Dennis ES, Brettell RIS, Peacock WJ (1987) A tissue culture induced Adh1 null mutant of maize results from a single base change. Mol Gen Genet 210:181–183

    Article  CAS  Google Scholar 

  • Endo M, Kumagai M, Motoyama R, Sasaki-Yamagata H, Mori-Hosokawa S, Hamada M, Kanamori H, Nagamura Y, Katayose Y, Itoh T, Toki S (2015) Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting. Plant Cell Physiol 56:116–125

    Article  CAS  PubMed  Google Scholar 

  • Fang SC, Chen JC, Wei MJ (2016) Protocorms and protocorm-like bodies are molecularly distinct from zygotic embryonic tissues in Phalaenopsis aphrodite. Plant Physiol 171:2682–2700

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gao DY, Vallejo VA, He B, Gai YC, Sun LH (2009) Detection of DNA changes in somaclonal mutants of rice using SSR markers and transposon display. Plant Cell Tissue Organ Cult 98:187–196

    Article  CAS  Google Scholar 

  • Hang A, Bregitzer P (1993) Chromosomal variations in immature embryo-derived calli from six barley cultivars. J Hered 84:105–108

    Article  Google Scholar 

  • Hirochika H (1993) Activation of tobacco retrotransposons during tissue culture. EMBO J 12:2521–2528

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hsu TW, Tsai WC, Wang DP, Lin S, Hsiao YY, Chen WH, Chen HH (2008) Differential gene expression analysis by cDNA-AFLP between flower buds of Phalaenopsis Hsiang Fei cv. H. F. and its somaclonal variant. Plant Sci 175:415–422

    Article  CAS  Google Scholar 

  • Huan LVT, Takamura T, Tanaka M (2004) Callus formation and plant regeneration from callus through somatic embryo structures in Cymbidium orchid. Plant Sci 166:1443–1449

    Article  CAS  Google Scholar 

  • Jeong IS, Yoon UH, Lee GS, Ji HS, Lee HJ, Han CD, Hahn JH, An G, Kim TH (2013) SNP-based analysis of genetic diversity in anther-derived rice by whole genome sequencing. Rice 6:6

    Article  PubMed  PubMed Central  Google Scholar 

  • Jheng FY, Do YY, Liauh YW, Chung JP, Huang PL (2006) Enhancement of growth and regeneration efficiency from embryogenic callus cultures of Oncidium ‘Gower Ramsey’ by adjusting carbohydrate sources. Plant Sci 170:1133–1140

    Article  CAS  Google Scholar 

  • Jiang C, Mithani A, Gan X, Belfield EJ, Klingler JP, Zhu JK, Ragoussis J, Mott R, Harberd NP (2011) Regenerant Arabidopsis lineages display a distinct genome-wide spectrum of mutations conferring variant phenotypes. Curr Biol 21:1385–1390

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jiang W, Hua S, Zhou X, Han P, Lu Q, Qiu Y (2018) Assessment of genetic stability and analysis of alkaloids potential in micropropagated plants of Croomia japonica Miquel, an endangered, medicinal plant in China and Japan. Plant Cell Tissue Organ Cult 135:1–12

    Article  CAS  Google Scholar 

  • Kashima K, Mejima M, Kurokawa S, Kuroda M, Kiyono H, Yuki Y (2015) Comparative whole-genome analyses of selection marker-free rice-based cholera toxin B-subunit vaccine lines and wild-type lines. BMC Genom 16:48

    Article  CAS  Google Scholar 

  • Keerio AA, Shen C, Nie Y, Ahmed MM, Zhang X, Lin Z (2018) QTL mapping for fiber quality and yield traits based on introgression lines derived from Gossypium hirsutum × G. tomentosum. Int J Mol Sci 19:243

    Article  CAS  PubMed Central  Google Scholar 

  • Kent WJ (2002) BLAT—the BLAST-like alignment tool. Genome Res 12:656–664

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kiselev KV, Shumakova OA, Tchernoded GK (2011) Mutation of Panax ginseng genes during long-term cultivation of ginseng cell cultures. J Plant Physiol 168:1280–1285

    Article  CAS  PubMed  Google Scholar 

  • Kovalchuk I, Kovalchuk O, Hohn B (2000) Genome-wide variation of the somatic mutation frequency in transgenic plants. EMBO J 19:4431–4438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Krishna H, Alizadeh M, Singh D, Singh U, Chauhan N, Eftekhari M, Sadh RK (2016) Somaclonal variations and their applications in horticultural crops improvement. 3 Biotech 6:54

    Article  PubMed  PubMed Central  Google Scholar 

  • Larkin PJ, Scowcroft WR (1981) Somaclonal variation — a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214

    Article  CAS  PubMed  Google Scholar 

  • Larkin PJ, Banks PM, Bhati R, Brettell RIS, Davies PA, Ryan SA, Scowcroft WR, Spindler LH, Tanner GJ (1989) From somatic variation to variant plants: mechanisms and applications. Genome 31:705–711

    Article  Google Scholar 

  • Lee YI, Hsu ST, Yeung EC (2013) Orchid protocorm-like bodies are somatic embryos. Am J Bot 100:2121–2131

    Article  CAS  PubMed  Google Scholar 

  • Leshem B (1986) Carnation plantlets from vitrified plants as a source of somaclonal variation. HortScience 21:320–321

    Google Scholar 

  • Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Proc GPD (2009) The sequence alignment/map format and SAM tools. Bioinformatics 25:2078–2079

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li R, Qu R, Bruneau AH, Livingston DP (2010) Selection for freezing tolerance in St. Augustinegrass through somaclonal variation and germplasm evaluation. Plant Breed 129:417–421

    Article  Google Scholar 

  • Ling DH, Ma ZR, Chen WY, Chen MF (1987) Male sterile mutant from somatic cell culture of rice. Theor Appl Genet 75:127–131

    Article  Google Scholar 

  • Long H, Sung W, Miller SF, Ackerman MS, Doak TG, Lynch M (2014) Mutation rate, spectrum, topology, and context-dependency in the DNA mismatch repair-deficient Pseudomonas fluorescens ATCC948. Genome Biol Evol 7:262–271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Machczyńska J, Zimny J, Bednarek PT (2015) Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants. Plant Mol Biol 89:279–292

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miguel C, Marum L (2011) An epigenetic view of plant cells cultured in vitro: somaclonal variation and beyond. J Exp Bot 62:3713–3725

    Article  CAS  PubMed  Google Scholar 

  • Miyao A, Nakagome M, Ohnuma T, Yamagata H, Kanamori H, Katayose Y, Takahashi A, Matsumoto T, Hirochika H (2012) Molecular spectrum of somaclonal variation in regenerated rice revealed by whole-genome sequencing. Plant Cell Physiol 53:256–264

    Article  CAS  PubMed  Google Scholar 

  • Morel GM (1960) Producing virus-free Cymbidium. Am Orchid Soc Bull 29:495–497

    Google Scholar 

  • Müller E, Brown PT, Hartke S, Lörz H (1990) DNA variation in tissue-culture-derived rice plants. Theor Appl Genet 80:673–679

    Article  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Noro Y, Takanoshimizu T, Syono K, Kishima Y, Sano Y (2007) Genetic variations in rice in vitro cultures at the EPSPs–RPS20 region. Theor Appl Genet 114:705–711

    Article  CAS  PubMed  Google Scholar 

  • Okamoto H, Hirochika H (2010) Efficient insertion mutagenesis of Arabidopsis by tissue culture-induced activation of the tobacco retrotransposon Tto1. Plant J 23:291–304

    Article  Google Scholar 

  • Ossowski S, Schneeberger K, Lucas-Lledó JI, Warthmann N, Clark RM, Shaw RG, Weigel D, Lynch M (2010) The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana. Science 327:92–94

    Article  CAS  PubMed  Google Scholar 

  • Palombi MA, Lombardo B, Caboni E (2007) In vitro regeneration of wild pear (Pyrus pyraster Burgsd) clones tolerant to Fe-chlorosis and somaclonal variation analysis by RAPD markers. Plant Cell Rep 26:489–496

    Article  CAS  PubMed  Google Scholar 

  • Peng X, Zhang T, Zhang J (2015) Effect of subculture times on genetic fidelity, endogenous hormone level and pharmaceutical potential of Tetrastigma hemsleyanum callus. Plant Cell Tissue Organ Cult 122:67–77

    Article  CAS  Google Scholar 

  • Peschke VM, Phillips RL, Gengenbach BG (1987) Discovery of transposable element activity among progeny of tissue culture-derived maize plants. Science 238:804–807

    Article  CAS  PubMed  Google Scholar 

  • Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, pp 365–386

    Google Scholar 

  • Slazak B, Sliwinska E, Saługa M, Ronikier M, Bujak J, Słomka A, Göransson U, Kuta E (2015) Micropropagation of Viola uliginosa (Violaceae) for endangered species conservation and for somaclonal variation-enhanced cyclotide biosynthesis. Plant Cell Tissue Organ Cult 120:179–190

    Article  CAS  Google Scholar 

  • Su J, Pang C, Wei H, Li L, Liang B, Wang C, Song M, Wang H, Zhao S, Jia X, Mao G, Huang L, Geng D, Wang C, Fan S, Yu S (2016) Identification of favorable SNP alleles and candidate genes for traits related to early maturity via GWAS in upland cotton. BMC Genom 17:687

    Article  CAS  Google Scholar 

  • Sun X, Liu D, Zhang X, Li W, Liu H, Hong W, Jiang C, Guan N, Ma C, Zeng H, Xu C, Song J, Huang L, Wang C, Shi J, Wang R, Zheng X, Lu C, Wang X, Zheng H (2013) SLAF-seq: an efficient method of large-scale de novo SNP discovery and genotyping using high-throughput sequencing. PLoS ONE 8:e58700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Viehmannova I, Cepkova PH, Vitamvas J, Streblova P, Kisilova J (2016) Micropropagation of a giant ornamental bromeliad Puya berteroniana through adventitious shoots and assessment of their genetic stability through ISSR primers and flow cytometry. Plant Cell Tissue Organ Cult 125:293–302

    Article  CAS  Google Scholar 

  • Wang CX, Tian M (2014) Callus-mediated and direct protocorm-like body formation of Bletilla striata and assessment of clonal fidelity using ISSR markers. Acta Physiol Plant 36:2321–2330

    Article  CAS  Google Scholar 

  • Wang QM, Wang L (2012) An evolutionary view of plant tissue culture: somaclonal variation and selection. Plant Cell Rep 31:1535–1547

    Article  CAS  PubMed  Google Scholar 

  • Zhang D, Wang Z, Wang N, Gao Y, Liu Y, Wu Y, Bai Y, Zhang Z, Lin X, Dong Y, Ou X, Xu C, Liu B (2014) Tissue culture-induced heritable genomic variation in rice, and their phenotypic implications. PLoS One 9:e96879

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang J, Zhang Q, Cheng T, Yang W, Pan H, Zhong J, Huang L, Liu E (2015) High-density genetic map construction and identification of a locus controlling weeping trait in an ornamental woody plant (Prunus mume Sieb. et Zucc). DNA Res 22:183–191

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhao P, Wu F, Feng FS, Wang WJ (2008) Protocorm-like body (PLB) formation and plant regeneration from the callus culture of Dendrobium candidum Wall ex Lindl. In Vitro Cell Dev Biol Plant 44:178–185

    Article  CAS  Google Scholar 

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Acknowledgements

This study was funded by the National Natural Science Foundation of China (Grant No. 31301801) and the Fundamental Research Funds for the Central Non-profit Research Institution of CAF (Grant No. CAFYBB2014QB012). The authors are grateful for the assistance provided by Biomarker Technologies (Beijing, China) in data processing.

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Authors and Affiliations

  1. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, 311400, Hangzhou, China

    Caixia Wang, Min Tian, Ying Zhang & Tong Ouyang

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  1. Caixia Wang
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  2. Min Tian
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  3. Ying Zhang
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  4. Tong Ouyang
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Contributions

CXW and MT conceived the study and designed the experiments. CXW performed the experiments, analyzed the data and wrote the manuscript. MT revised the manuscript. YZ and TO helped in culturing the plant materials. All authors read and approved the final manuscript.

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Correspondence to Min Tian.

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Communicated by Ming-Tsair Chan.

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Wang, C., Tian, M., Zhang, Y. et al. Molecular spectrum of somaclonal variation in PLB-regenerated Oncidium revealed by SLAF-seq. Plant Cell Tiss Organ Cult 137, 541–552 (2019). https://doi.org/10.1007/s11240-019-01589-4

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