Comparative transcriptomics analysis uncovers alternative splicing events and molecular markers in cabbage (Brassica oleracea L.)

Abstract

Main conclusion

Alternative splicing (AS) events were identified and verified in cabbage by comparative transcriptome analysis. The corresponding markers were developed and the germplasm resources were identified.

Alternative splicing (AS) is a central regulatory mechanism that greatly contributes to plant gene expression and transcriptome diversity. A large body of evidence has shown that AS complexity is relevant for plant development, evolution, complexity, and adaptation. Both insertion/deletion (InDel) and single nucleotide polymorphism (SNP) are typically co-dominant inheritance markers and have abundant polymorphisms. These have been widely used for marker-assisted selection, genetic mapping, and germplasm identification in plants. However, little is known about the molecular mechanisms underlying AS events and the development of markers including SNP and InDel from the cabbage transcriptome. In this study, three cabbage transcriptome datasets were collected and aligned to the cabbage reference genome to analyze AS events and marker development. 31,524 AS events were identified from three cabbage genotypes, accounting for 20.8% of the total cabbage genes. Alternative 3′ splice site donor (A3SS) was the most frequent type of the four main AS events in cabbage. 70,475 InDels and 706,269 SNPs were identified with average frequencies of 1 InDel/6.9 kb and 1 SNP/0.7 kb, respectively. 71,942 potential SSRs were identified in 53,129 assembled unigenes with a density of 1 SSR/6.8 kb. The ratio of SNPs with synonymous/non-synonymous mutations was 1:0.65. 142 InDels and 36 SNPs were randomly selected and validated via Sanger sequencing and polymorphism was found among 66.2% of the InDels and 78.6% of the SNPs. Furthermore, 35 informative InDel markers were successfully used for genetic diversity analysis on 36 cabbage accessions. These results facilitate understanding of the molecular regulation mechanism underlying AS events in cabbage. They also provide molecular marker resource data for genetic mapping construction and germplasm identification, and facilitate the genetic improvement of cabbage via breeding.

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Abbreviations

AS:

Alternative splicing

A3SS:

Alternative 3′ splice site donor

A5SS:

Alternative 5′ splice site donor

CDS:

Coding sequence

ES:

Exon skipping

HE:

Expected heterozygosity

HO:

Expected homozygosity

InDel:

Insertion/deletion

IR:

Intron retention

Na:

Number of alleles

NCBI:

National center for biotechnology information

PAGE:

Polyacrylamide gel electrophoresis

PIC:

Polymorphic information content

SNP:

Single nucleotide polymorphism

SSR:

Simple sequence repeat

Ts:

Transition

Tv:

Transversion

UPGMA:

Unweighted pair-group method with arithmetic averages

References

  1. Blencowe BJ (2006) Alternative splicing: new insights from global analyses. Cell 126:37–47

    CAS  PubMed  Google Scholar 

  2. Cingolani P, Platts A, Wan LL, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6:80–92

    CAS  Google Scholar 

  3. Clark RM, Schweikert G, Toomajian C, Ossowski S, Zeller G, Shinn P, Warthmann N, Hu TT, Fu G, Hinds DA, Chen H, Frazer KA, Huson DH, Schölkopf B, Nordborg M, Rätsch G, Ecker JR, Weigel D (2007) Common sequence polymorphisms shaping genetic diversity in Arabidopsis thaliana. Science 317:338–342

    CAS  PubMed  Google Scholar 

  4. van Dijk EL, Auger H, Jaszczyszyn Y, Thermes C (2014) Ten years of next-generation sequencing technology. Trends Genet 30:418–426

    PubMed  Google Scholar 

  5. Filichkin SA, Priest HD, Givan SA, Shen R, Bryant DW, Fox SE, Wong WK, Mockler TC (2010) Genome-wide mapping of alternative splicing in Arabidopsis thaliana. Genome Res 20:45–58

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Foissac S, Sammeth M (2007) ASTALAVISTA: dynamic and flexible analysis of alternative splicing events in custom gene datasets. Nucleic Acids Res 35:W297–299

    PubMed  PubMed Central  Google Scholar 

  7. García-Lor A, Luro F, Navarro L, Ollitrault P (2012) Comparative use of InDel and SSR markers in deciphering the interspecific structure of cultivated citrus genetic diversity: a perspective for genetic association studies. Mol Gen Genomics 287:77–94

    Google Scholar 

  8. Guo Y, Wiegert-Rininger KE, Vallejo VA, Barry CS, Warner RM (2015) Transcriptome-enabled marker discovery and mapping of plastochron-related genes in Petunia spp. BMC Genom 16:1

    Google Scholar 

  9. Guo Y, Su B, Tang J, Zhou F, Qiu LJ (2017) Gene-based SNP identification and validation in soybean using next-generation transcriptome sequencing. Mol Genet Genomics 293:623–633

    PubMed  Google Scholar 

  10. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, Macmanes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, Leduc RD, Friedman N, Regev A (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512

    CAS  PubMed  Google Scholar 

  11. Huang J, Gao Y, Jia H, Liu L, Zhang D, Zhang Z (2015) Comparative transcriptomics uncovers alternative splicing changes and signatures of selection from maize improvement. BMC Genom 16:363

    Google Scholar 

  12. Izzah NK, Lee J, Jayakodi M, Perumal S, Jin M, Park BS, Ahn K, Yang TJ (2014) Transcriptome sequencing of two parental lines of cabbage (Brassica oleracea L. var. capitata L.) and construction of an EST-based genetic map. BMC Genom 15:149

    PubMed  PubMed Central  Google Scholar 

  13. Katz Y, Wang ET, Airoldi EM, Burge CB (2010) Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat Methods 7:1009–1015

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Kawamura K, Shimizu M, Kawanabe T, Pu Z, Kodama T, Kaji M, Osabe K, Fujimoto R, Okazaki K (2017) Assessment of DNA markers for seed contamination testing and selection of disease resistance in cabbage. Euphytica 213:28

    Google Scholar 

  15. Keller I, Bensasson D, Nichols RA (2007) Transition-transversion bias is not universal: a counter example from grasshopper pseudogenes. PLoS Genet 3:e22

    PubMed  PubMed Central  Google Scholar 

  16. Kim HA, Lim CJ, Kim S, Choe JK, Jo SH, Baek N, Kwon SY (2014) High-Throughput sequencing and de novo assembly of Brassica oleracea var. Capitata L. for transcriptome analysis. Plos One 9:e92087

    PubMed  PubMed Central  Google Scholar 

  17. Kim SR, Ramos J, Ashikari M, Virk PS, Torres EA, Nissila E, Hechanova SL, Mauleon R, Jena KK (2016) Development and validation of allele-specific SNP/indel markers for eight yield-enhancing genes using whole-genome sequencing strategy to increase yield potential of rice, oryza sativa L. Rice 9:12

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Lee J, Izzah NK, Jayakodi M, Perumal S, Joh HJ, Lee HJ, Lee SC, Park JY, Yang KW, Nou IS, Seo J, Yoo J, Suh Y, Ahn K, Lee JH, Choi GJ, Yu Y, Kim H, Yang TJ (2015) Genome-wide SNP identification and QTL mapping for black rot resistance in cabbage. BMC Plant Biol 15:32

    PubMed  PubMed Central  Google Scholar 

  19. Lee J, Izzah NK, Choi BS, Joh HJ, Lee SC, Perumal S, Seo J, Ahn K, Jo EJ, Choi GJ, Nou IS, Yu Y, Yang TJ (2016) Genotyping-by-sequencing map permits identification of clubroot resistance QTLs and revision of the reference genome assembly in cabbage (Brassica oleracea L.). DNA Res 23:39–41

    Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  21. Li D, Zeng R, Li Y, Zhao M, Chao J, Li Y, Wang K, Zhu L, Tian WM, Liang C (2016) Gene expression analysis and SNP/InDel discovery to investigate yield heterosis of two rubber tree F1 hybrids. Sci Rep 6:24984

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Liu K, Muse SV (2005) PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics 21:2128–2129

    CAS  PubMed  Google Scholar 

  23. Liu S, Liu Y, Yang X, Tong C, Edwards D, Parkin IA, Zhao M, Ma J, Yu J, Huang S, Wang X, Wang J, Lu K, Fang Z, Bancroft I, Yang TJ, Hu Q, Wang X, Yue Z, Li H, Yang L, Wu J, Zhou Q, Wang W, King GJ, Pires JC, Lu C, Wu Z, Sampath P, Wang Z, Guo H, Pan S, Yang L, Min J, Zhang D, Jin D, Li W, Belcram H, Tu J, Guan M, Qi C, Du D, Li J, Jiang L, Batley J, Sharpe AG, Park BS, Ruperao P, Cheng F, Waminal NE, Huang Y, Dong C, Wang L, Li J, Hu Z, Zhuang M, Huang Y, Huang J, Shi J, Mei D, Liu J, Lee TH, Wang J, Jin H, Li Z, Li X, Zhang J, Xiao L, Zhou Y, Liu Z, Liu X, Qin R, Tang X, Liu W, Wang Y, Zhang Y, Lee J, Kim HH, Denoeud F, Xu X, Liang X, Hua W, Wang X, Wang J, Chalhoub B, Paterson AH (2011) The brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat Commun 5:3930

    Google Scholar 

  24. Liu B, Wang Y, Zhai W, Deng J, Wang H, Cui Y, Cheng F, Wang X, Wu J (2013) Development of InDel markers for Brassica rapa based on whole-genome re-sequencing. Theor Appl Genet 126:231–239

    CAS  PubMed  Google Scholar 

  25. Liu J, Chen X, Liang X, Zhou X, Yang F, Liu J, He SY, Guo Z (2016) Alternative splicing of rice WRKY62 and WRKY76 transcription factor genes in pathogen defense. Plant Physiol 171:1427–1442

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Lu J, Bi H, Zhang A, Guo T, Li Y, Li Y (2018) Comparative transcriptome analysis by RNA-Seq of the regulation of low temperature responses in Dendranthema morifolium. Hortic Environ Biotechnol 59:1–13

    Google Scholar 

  27. Luo X, Xu L, Liang D, Wang Y, Zhang W, Zhu X, Zhu Y, Jiang H, Tang M, Liu L (2017) Comparative transcriptomics uncovers alternative splicing and molecular marker development in radish (Raphanus sativus L.). BMC Genom 18:505

  28. Lv H, Fang Z, Yang L, Zhang Y, Wang Q, Liu Y, Zhuang M, Yang Y, Xie B, Liu B, Liu J, Kang J, Wang X (2014) Mapping and analysis of a novel candidate Fusarium wilt resistance gene FOC1 in Brassica oleracea. BMC Genom 15:1094

    Google Scholar 

  29. Mandadi KK, Scholthof KB (2015) Genome-wide analysis of alternative splicing landscapes modulated during plant-virus interactions in Brachypodium distachyon. Plant Cell 27:71–85

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Marquez Y, Brown JW, Simpson C, Barta A, Kalyna M (2012) Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. Genome Res 22:1184–1195

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 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

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Merkin J, Russell C, Chen P, Burge CB (2012) Evolutionary dynamics of gene and isoform regulation in mammalian tissues. Science 338:1593–1599

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Min XJ, Powell B, Braessler J, Meinken J, Yu F, Sablok G (2015) Genome-wide cataloging and analysis of alternatively spliced genes in cereal crops. BMC Genom 16:721

    Google Scholar 

  34. Mun JH, Chung H, Chung WH, Oh M, Jeong YM, Kim N, Ahn BO, Park BS, Park S, Lim KB, Hwang YJ, Yu HJ (2015) Construction of a reference genetic map of Raphanus sativus based on genotyping by whole-genome resequencing. Theor Appl Genet 128:259–272

    CAS  PubMed  Google Scholar 

  35. Parkin IAP, Koh C, Tang H, Robinson SJ, Kagale S, Clarke WE, Town CD, Nixon J, Krishnakumar V, Bidwell SL, Denoeud F, Belcram H, Links MG, Just J, Clarke C, Bender T, Huebert T, Mason AS, Pires JC, Barker G, Moore J, Walley PG, Manoli S, Batley J, Edwards D, Nelson MN, Wang X, Paterson AH, King G, Bancroft L, Chalhoub B, Sharpe AG (2014) Transcriptome and methylome profiling reveals relics of genome dominance in the mesopolyploid Brassica oleracea. Genome Biol 15:R77

    PubMed  PubMed Central  Google Scholar 

  36. Qu C, Jia L, Fu F, Zhao H, Lu K, Wei L, Xu X, Liang Y, Li S, Wang R, Li J (2017) Genome-wide association mapping and identification of candidate genes for fatty acid composition in Brassica napus L. using SNP markers. BMC Genom 18:232

  37. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Rodríguez SA, Grochová D, McKenna T, Borate B, Trivedi NS, Erdos MR, Eriksson M (2016) Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging. Aging Cell 15:267–278

    PubMed  Google Scholar 

  39. Ruan J, Guo F, Wang Y, Li X, Wan S, Shan L, Peng Z (2018) Transcriptome analysis of alternative splicing in peanut (Arachis hypogaea L.). BMC Plant Biol 18:139

  40. Shen S, Park JW, Lu ZX, Lin L, Henry MD, Wu YN, Zhou Q, Xing Y (2014a) rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data. Proc Natl Acad Sci U S A 111:E5593–5601

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Shen Y, Zhou Z, Wang Z, Li W, Fang C, Wu M, Ma Y, Liu T, Kong LA, Peng DL, Tian Z (2014b) Global dissection of alternative splicing in paleopolyploid soybean. Plant Cell 26:996–1008

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Shi L, Chen X, Chen W, Zheng Y, Yang Z (2018) Comparative transcriptomic analysis of white and red chinese bayberry (Myrica rubra) fruits reveals flavonoid biosynthesis regulation. Sci Hort 235:9–20

    Google Scholar 

  43. Souza LMD, Toledo-Silva G, Cardoso-Silva CB, Silva CCD, Andreotti IADA, Conson ARO, Mantello CC, Guen VL, Souza APD (2016) Development of single nucleotide polymorphism markers in the large and complex rubber tree genome using next-generation sequence data. Mol Breed 36:115

    Google Scholar 

  44. Su T, Li P, Yang J, Sui G, Yu Y, Zhang D, Zhao X, Wang W, Wen C, Yu S, Zhang F (2018) Development of cost-effective single nucleotide polymorphism marker assays for genetic diversity analysis in Brassica rapa. Mol Breed 38:42

    Google Scholar 

  45. Sun Y, Xiao H (2015) Identification of alternative splicing events by RNA sequencing in early growth tomato fruits. BMC Genom 16:948

    Google Scholar 

  46. Taheri S, Lee Abdullah T, Yusop MR, Hanafi MM, Sahebi M, Azizi P, Shamshiri RR (2018) Mining and development of novel SSR markers using Next Generation Sequencing (NGS) data in plants. Molecules 23:1–20

    Google Scholar 

  47. Thatcher SR, Zhou W, Leonard A, Wang BB, Beatty M, Zastrow-Hayes G, Zhao X, Baumgarten A, Li B (2014) Genome-wide analysis of alternative splicing in Zea mays: landscape and genetic regulation. Plant Cell 26:3472–3487

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Thatcher SR, Danilevskaya ON, Meng X, Beatty M, Zastrow-Hayes G, Harris C, Van Allen B, HabbenJ Li B (2016) Genome-wide analysis of alternative splicing during development and drought stress in maize. Plant Physiol 170:586–599

    CAS  PubMed  Google Scholar 

  49. Thiel T, Michalek W, Varshney RK, Graner A (2003) Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet 106:411–422

    CAS  PubMed  Google Scholar 

  50. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105–1111

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562–578

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Vitulo N, Forcato C, Carpinelli EC, Telatin A, Campagna D, D'Angelo M, Zimbello R, Corso M, Vannozzi A, Bonghi C, Lucchin M, Valle G (2014) A deep survey of alternative splicing in grape reveals changes in the splicing machinery related to tissue, stress condition and genotype. BMC Plant Biol 14:99

    PubMed  PubMed Central  Google Scholar 

  53. Wang BB, Brendel V (2006) Genome wide comparative analysis of alternative splicing in plants. Proc Natl Acad Sci U S A 103:7175–7180

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Wang Y, Liu W, Xu L, Wang Y, Chen Y, Luo X, Tang M, Liu L (2017) Development of SNP markers based on transcriptome sequences and their application in germplasm identification in radish (Raphanus sativus L.) Mol Breed 37:26

  55. Wang J, Kong L, Yu K, Zhang F, Shi X, Wang Y, Nan H, Zhao X, Lu S, Cao D, Li X, Fang C, Wang F, Su T, Li S, Yuan X, Liu B, Kong F (2018) Development and validation of InDel markers for identification of QTL underlying flowering time in soybean. Crop J 6:126–135

    CAS  Google Scholar 

  56. Wei L, Miao H, Li C, Duan Y, Niu J, Zhang T, Zhao Q, Zhang H (2014) Development of SNP and InDel markers via de novo transcriptome assembly in Sesamum indicum L. Mol Breed 34:2205–2217

    CAS  Google Scholar 

  57. Xiao Z, Hu Y, Zhang X, Xue Y, Fang Z, Yang L, Zhang Y, Liu Y, Li Z, Liu X, Liu Z, Lv H, Zhuang M (2017) Fine mapping and transcriptome analysis reveal candidate genes associated with hybrid lethality in cabbage (Brassica oleracea). Genes 8:2–17

    Google Scholar 

  58. Xu X, Liu X, Ge S, Jensen JD, Hu F, Li X, Dong Y, Gutenkunst RN, Fang L, Huang L, Li J, He W, Zhang G, Zheng X, Zhang F, Li Y, Yu C, Kristiansen K, Zhang X, Wang J, Wright M, McCouch S, Nielsen R, Wang J, Wang W (2012) Resequencing 50 accessions of cultivated and wild rice yields markers for identifying agronomically important genes. Nat Biotechnol 30:105–111

    CAS  Google Scholar 

  59. Yang J, Wang Y, Shen H, Yang W (2014a) In Silico identification and experimental validation of Insertion-Deletion polymorphisms in tomato genome. DNA Res 21:429–438

    PubMed  PubMed Central  Google Scholar 

  60. Yang S, Tang F, Zhu H (2014b) Alternative splicing in plant immunity. Int J Mol Sci 15:10424–10445

    PubMed  PubMed Central  Google Scholar 

  61. Yang H, Li C, Lam HM, Clements J, Yan G, Zhao S (2015) Sequencing consolidates molecular markers with plant breeding practice. Theor Appl Genet 128:779–795

    CAS  PubMed  Google Scholar 

  62. Yang Z, Dai Z, Xie D, Chen J, Tang Q, Cheng C, Xu Y, Wang T, Su J (2018) Development of an InDel polymorphism database for jute via comparative transcriptome analysis. Genome 61:323–327

    CAS  PubMed  Google Scholar 

  63. Zhang JZ, Liu SR, Hu CG (2016a) Identifying the genome-wide genetic variation between precocious trifoliate orange and its wild type and developing new markers for genetics research. DNA Res 23:403–414

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Zhang Q, Zhang X, Wang S, Tan C, Zhou G, Li C (2016b) Involvement of alternative splicing in barley seed germination. PLoS ONE 11:e0152824

    PubMed  PubMed Central  Google Scholar 

  65. Zhang X, Liu Y, Fang Z, Li Z, Yang L, Zhuang M, Zhang Y, Lv H (2016c) Comparative transcriptome analysis between Broccoli (Brassica oleracea var. italica) and Wild Cabbage (Brassica macrocarpa Guss.) in Response to Plasmodiophora brassicae during different infection stages. Front Plant Sci 7:1929

  66. Zhang T, Gu M, Liu Y, Lv Y, Zhou L, Lu H, Liang S, Bao H, Zhao H (2017) Development of novel inDel markers and genetic diversity in chenopodium quinoa through whole-genome re-sequencing. BMC Genom 18:685

    Google Scholar 

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Acknowledgments

This work was in part supported by grants from the National Key Research and Development Program of China (2016YFD0100204).

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Correspondence to Aisong Zeng or Jiyong Yan.

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Xu, Y., Zeng, A., Song, L. et al. Comparative transcriptomics analysis uncovers alternative splicing events and molecular markers in cabbage (Brassica oleracea L.). Planta 249, 1599–1615 (2019). https://doi.org/10.1007/s00425-019-03108-3

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Keywords

  • Cabbage
  • Transcriptome
  • Genetic diversity
  • Single-nucleotide polymorphism (SNP)
  • Alternative splicing (AS)
  • Insertion/deletion (InDel)