TILLING by Sequencing (TbyS) for targeted genome mutagenesis in crops

Molecular Breeding

Volumn 37, Issue 2, 2017, Pages

  Kumar, Anishkumar P K ^a,b   McKeown, Peter C ^a   Boualem, Adnane ^c   Ryder, Peter ^a   Brychkova, Galina ^a   Bendahmane, Abdelhafid ^c   Sarkar, Abhimanyu ^d   Chatterjee, Manash ^a,b   Spillane, Charles ^a  

^a NATIONAL UNIVERSITY OF IRELAND   (Ireland)

^b Bench Bio PvtLtd   (India)

^c INSTITUTE OF PLANT SCIENCES PARIS SACLAY IPS2   (France)

^d JOHN INNES CENTRE   (United Kingdom)

Author keywords

CRISPR Cas9; Genome editing; Induced variation; Mutagenesis; TbyS; TILLING by Sequencing

Indexed keywords

GENES; MUTAGENESIS;

BREEDING PROGRAMMES; CRISPR/CAS9; FUNCTIONAL GENOMICS; GENE CONTROLLING; GENETIC VARIATION; GENOME EDITING; HIGH-THROUGHPUT SEQUENCING; INDUCED VARIATION; TARGETING INDUCED LOCAL LESION IN GENOME BY SEQUENCING; TILLING BY SEQUENCING;

CROPS;

EID: 85011661369     PISSN: 13803743     EISSN: 15729788     Source Type: Journal    
DOI: 10.1007/s11032-017-0620-1     Document Type: Review

References (104)

1. Abbott A (2015) Europe’s genetically edited plants stuck in legal limbo. Nature 528:319–320
2. Acevedo-Garcia J, Spencer D, Thieron H, Reinstadler A, Hammond-Kosack K, Phillips AL, Panstruga R (2016) mlo-based powdery mildew resistance in hexaploid bread wheat generated by a non-transgenic TILLING approach. Plant Biotechnol J. doi:10.1111/pbi.12631
3. Alagoz Y, Gurkok T, Zhang B, Unver T (2016) Manipulating the biosynthesis of bioactive compound alkaloids for next-generation metabolic engineering in opium poppy using CRISPR-Cas 9 genome editing technology. Sci Rep 6:30910. doi:10.1038/srep30910
4. Basak J, Nithin C (2015) Targeting non-coding RNAs in plants with the CRISPR-Cas technology is a challenge yet worth accepting. frontiers in plant science 6
5. Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9(1):39
6. Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V (2015) Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol 32:76–84. doi:10.1016/j.copbio.2014.11.007
7. Berbel A, Ferrandiz C, Hecht V, Dalmais M, Lund OS, Sussmilch FC, Taylor SA, Bendahmane A, Ellis TH, Beltran JP, Weller JL, Madueno F (2012) VEGETATIVE1 is essential for development of the compound inflorescence in pea. Nat Commun 3:797. doi:10.1038/ncomms1801
8. Blomstedt CK, Gleadow RM, O'Donnell N, Naur P, Jensen K, Laursen T, Olsen CE, Stuart P, Hamill JD, Moller BL, Neale AD (2012) A combined biochemical screen and TILLING approach identifies mutations in Sorghum bicolor L. Moench resulting in acyanogenic forage production. Plant Biotechnol J 10(1):54–66. doi:10.1111/j.1467-7652.2011.00646.x
9. Boualem A, Fleurier S, Troadec C, Audigier P, Kumar AP, Chatterjee M, Alsadon AA, Sadder MT, Wahb-Allah MA, Al-Doss AA (2014) Development of a Cucumis sativus TILLinG platform for forward and reverse genetics. PLoS One 9(5):e97963
10. Brooks C, Nekrasov V, Lippman ZB, Van Eck J (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166(3):1292–1297. doi:10.1104/pp.114.247577
11. Brozynska M, Furtado A, Henry RJ (2016) Genomics of crop wild relatives: expanding the gene pool for crop improvement. Plant Biotechnol J 14(4):1070–1085. doi:10.1111/pbi.12454
12. Cermak T, Baltes NJ, Cegan R, Zhang Y, Voytas DF (2015) High-frequency, precise modification of the tomato genome. Genome Biol 16(1):232. doi:10.1186/s13059-015-0796-9
13. Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, Pearlsman M, Sherman A, Arazi T, Gal-On A (2016) Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology. Molecular plant pathology
14. Comai L, Young K, Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Henikoff S (2004) Efficient discovery of DNA polymorphisms in natural populations by ecotilling. Plant J 37(5):778–786
15. Cooper HD, Spillane C, Hodgkin T (2001) Broadening the genetic base of crop production. CABI
16. Cooper JL, Henikoff S, Comai L, Till BJ (2013) TILLING and ecotilling for rice. Methods Mol Biol 956:39–56. doi:10.1007/978-1-62703-194-3_4
17. Dahmani-Mardas F, Troadec C, Boualem A, Leveˆque S, Alsadon AA, Aldoss AA, Dogimont C, Bendahmane A (2010) Engineering melon plants with improved fruit shelf life using the TILLING approach. PLoS One 5(12):e15776
18. Egelie KJ, Graff GD, Strand SP, Johansen B (2016) The emerging patent landscape of CRISPR-Cas gene editing technology. Nat Biotechnol 34(10):1025–1031
19. Elahi N, Duncan RW, Stasolla C (2015) Decreased seed oil production in FUSCA3 Brassica napus mutant plants. Plant Physiol Biochem 96:222–230. doi:10.1016/j.plaphy.2015.08.002
20. Fan D, Liu T, Li C, Jiao B, Li S, Hou Y, Luo K (2015) Efficient CRISPR/Cas9-mediated targeted mutagenesis in Populus in the first generation. Scientific reports 5
21. Fang Y, Tyler BM (2015) Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9. Mol Plant Pathol 17:127–139
22. Gauffier C, Lebaron C, Moretti A, Constant C, Moquet F, Bonnet G, Caranta C, Gallois JL (2016) A TILLING approach to generate broad-spectrum resistance to potyviruses in tomato is hampered by eIF4E gene redundancy. The Plant journal: for cell and molecular biology 85(6):717–729. doi:10.1111/tpj.13136
23. Gil-Humanes J, Wang Y, Liang Z, Shan Q, Ozuna CV, Sanchez-Leon S, Baltes NJ, Starker C, Barro F, Gao C, Voytas DF (2016) High efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. Plant J. doi:10.1111/tpj.13446
24. Godfray HC, Garnett T (2014) Food security and sustainable intensification. Philos Trans R Soc Lond Ser B Biol Sci 369(1639):20120273. doi:10.1098/rstb.2012.0273
25. Gottwald S, Bauer P, Komatsuda T, Lundqvist U, Stein N (2009) TILLING in the two-rowed barley cultivar ‘Barke’ reveals preferred sites of functional diversity in the gene HvHox1. BMC Research Notes 2(1):258
26. Gross BL, Olsen KM (2010) Genetic perspectives on crop domestication. Trends Plant Sci 15(9):529–537
27. Hartung F, Schiemann J (2014) Precise plant breeding using new genome editing techniques: opportunities, safety and regulation in the EU. Plant J 78(5):742–752. doi:10.1111/tpj.12413
28. Henry IM, Nagalakshmi U, Lieberman MC, Ngo KJ, Krasileva KV, Vasquez-Gross H, Akhunova A, Akhunov E, Dubcovsky J, Tai TH (2014) Efficient genome-wide detection and cataloging of EMS-induced mutations using exome capture and next-generation sequencing. Plant Cell 26(4):1382–1397
29. Hofer J, Turner L, Moreau C, Ambrose M, Isaac P, Butcher S, Weller J, Dupin A, Dalmais M, Le Signor C, Bendahmane A, Ellis N (2009) Tendril-less regulates tendril formation in pea leaves. Plant Cell 21(2):420–428. doi:10.1105/tpc.108.064071
30. Huang S, Weigel D, Beachy RN, Li J (2016) A proposed regulatory framework for genome-edited crops. Nat Genet 48(2):109
31. King R, Bird N, Ramirez-Gonzalez R, Coghill JA, Patil A, Hassani-Pak K, Uauy C, Phillips AL (2015) Mutation scanning in wheat by exon capture and next-generation sequencing. PLoS One 10(9):e0137549. doi:10.1371/journal.pone.0137549
32. Konzak CF, Nilan RA, Kleinhofs A (1976) Artificial mutagenesis as an aid in overcoming genetic vulnerability of crop plants. Basic Life Sci 8:163–177
33. Kovach MJ, McCouch SR (2008) Leveraging natural diversity: back through the bottleneck. Curr Opin Plant Biol 11(2):193–200. doi:10.1016/j.pbi.2007.12.006
34. Kumar AP, Boualem A, Bhattacharya A, Parikh S, Desai N, Zambelli A, Leon A, Chatterjee M, Bendahmane A (2013) SMART—sunflower mutant population and reverse genetic tool for crop improvement. BMC Plant Biol 13:38. doi:10.1186/1471-2229-13-38
35. Lai KS, Kaothien-Nakayama P, Iwano M, Takayama S (2012) A TILLING resource for functional genomics in Arabidopsis thaliana accession C24. Genes & Genetic Systems 87(5):291–297
36. Ledford H (2016) Titanic clash over CRISPR patents turns ugly. Nature 537(7621):460–461
37. Li J, Meng X, Zong Y, Chen K, Zhang H, Liu J, Li J, Gao C (2016) Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9. Nat Plants 2:16139. doi:10.1038/nplants.2016.139
38. Lowder LG, Zhang D, Baltes NJ, Paul JW 3rd, Tang X, Zheng X, Voytas DF, Hsieh TF, Zhang Y, Qi Y (2015) A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol 169(2):971–985. doi:10.1104/pp.15.00636
39. Mao Y, Zhang Z, Feng Z, Wei P, Zhang H, Botella JR, Zhu JK (2016) Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis. Plant Biotechnol J 14(2):519–532
40. McDougall P (2011) The cost and time involved in the discovery, development and authorisation of a new plant biotechnology derived trait. Phillips McDougall, United Kingdom
41. McKeown PC, Fort A, Duszynska D, Sulpice R, Spillane C (2013a) Emerging molecular mechanisms for biotechnological harnessing of heterosis in crops. Trends Biotechnol 31:549–551
42. McKeown PC, Keshavaiah C, Fort A, Tuteja R, Chatterjee M, Varshney RK, Spillane C (2013b) Genomics in agriculture and food processing. In: Panesar PS, Marwaha SS (eds) Biotechnology in agriculture and food processing: Opportunities and challenges. Taylor & Francis, CRC Press
43. Meyer RS, Purugganan MD (2013) Evolution of crop species: genetics of domestication and diversification. Nat Rev Genet 14(12):840–852
44. Minoia S, Boualem A, Marcel F, Troadec C, Quemener B, Cellini F, Petrozza A, Vigouroux J, Lahaye M, Carriero F, Bendahmane A (2016) Induced mutations in tomato SlExp1 alter cell wall metabolism and delay fruit softening. Plant Sci 242:195–202. doi:10.1016/j.plantsci.2015.07.001
45. Missirian V, Comai L, Filkov V (2011) Statistical mutation calling from sequenced overlapping DNA pools in TILLING experiments. BMC Bioinformatics 12:287. doi:10.1186/1471-2105-12-287
46. Moose SP, Mumm RH (2008) Molecular plant breeding as the foundation for 21st century crop improvement. Plant Physiol 147(3):969–977. doi:10.1104/pp.108.118232
47. Morineau C, Bellec Y, Tellier F, Gissot L, Kelemen Z, Nogue F, Faure JD (2016) Selective gene dosage by CRISPR-Cas9 genome editing in hexaploid Camelina sativa. Plant Biotechnol J. doi:10.1111/pbi.12671
48. Morris SH, Spillane C (2008) GM directive deficiencies in the European Union. EMBO Rep 9(6):500–504. doi:10.1038/embor.2008.94
49. Murphy D (2007) Plant breeding and biotechnology. Cambridge University Press, Cambridge, UK
50. Nieto C, Piron F, Dalmais M, Marco CF, Moriones E, Gómez-Guillamón ML, Truniger V, Gómez P, Garcia-Mas J, Aranda MA (2007) EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility. BMC Plant Biol 7(1):34
51. Onda Y, Mochida K (2016) Exploring genetic diversity in plants using high-throughput sequencing techniques. Curr Genomics 17(4):358–367. doi:10.2174/1389202917666160331202742
52. Parry MA, Madgwick PJ, Bayon C, Tearall K, Hernandez-Lopez A, Baudo M, Rakszegi M, Hamada W, Al-Yassin A, Ouabbou H (2009) Mutation discovery for crop improvement. J Exp Bot 60(10):2817–2825
53. Perry J, Brachmann A, Welham T, Binder A, Charpentier M, Groth M, Haage K, Markmann K, Wang TL, Parniske M (2009) TILLING in Lotus japonicus identified large allelic series for symbiosis genes and revealed a bias in functionally defective ethyl methanesulfonate alleles toward glycine replacements. Plant Physiol 151(3):1281–1291
54. Puchta H (2016) Applying CRISPR/Cas for genome engineering in plants: the best is yet to come. Curr Opin Plant Biol 36:1–8. doi:10.1016/j.pbi.2016.11.011
55. Pyott DE, Sheehan E, Molnar A (2016) Engineering of CRISPR/Cas9-mediated potyvirus resistance in transgene-free Arabidopsis plants. Mol Plant Pathol 17(8):1276–1288. doi:10.1111/mpp.12417
56. Quetier F (2016) The CRISPR-Cas9 technology: closer to the ultimate toolkit for targeted genome editing. Plant Sci 242:65–76. doi:10.1016/j.plantsci.2015.09.003
57. Reddy TV, Dwivedi S, Sharma NK (2012) Development of TILLING by sequencing platform towards enhanced leaf yield in tobacco. Ind Crop Prod 40:324–335
58. Ricroch A, Harwood W, Svobodová Z, Sági L, Hundleby P, Badea EM, Rosca I, Cruz G, Salema Fevereiro MP, Marfà Riera V (2015) Challenges facing European agriculture and possible biotechnological solutions. Crit Rev Biotechnol:1–9
59. Ricroch AE, Ammann K, Kuntz M (2016a) Editing EU legislation to fit plant genome editing. EMBO reports: e201643099
60. Ricroch AE, Ammann K, Kuntz M (2016b) Editing EU legislation to fit plant genome editing. EMBO Rep. doi:10.15252/embr.201643099
61. Scully ED, Gries T, Funnell-Harris DL, Xin Z, Kovacs FA, Vermerris W, Sattler SE (2015) Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum. J Integr Plant Biol. doi:10.1111/jipb.12375
62. Sestili F, Botticella E, Bedo Z, Phillips A, Lafiandra D (2010) Production of novel allelic variation for genes involved in starch biosynthesis through mutagenesis. Mol Breed 25(1):145–154. doi:10.1007/s11032-009-9314-7
63. Seth K, Harish (2016) Current status of potential applications of repurposed Cas9 for structural and functional genomics of plants. Biochem Biophys Res Commun. doi:10.1016/j.bbrc.2016.10.130
64. Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu J-L (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31(8):686–688
65. Shen L, Wang C, Fu Y, Wang J, Liu Q, Zhang X, Yan C, Qian Q, Wang K (2016) QTL editing confers opposing yield performance in different rice varieties. J Integr Plant Biol. doi:10.1111/jipb.12501
66. Sheridan C (2014) First CRISPR-Cas patent opens race to stake out intellectual property. Nat Biotechnol 32(7):599–601. doi:10.1038/nbt0714-599
67. Sherkow JS (2015) Law, history and lessons in the CRISPR patent conflict. Nat Biotechnol 33(3):256–257. doi:10.1038/nbt.3160
68. Shi J, Gao H, Wang H, Lafitte HR, Archibald RL, Yang M, Hakimi SM, Mo H, Habben JE (2016) ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnol J. doi:10.1111/pbi.12603
69. Slade AJ, Fuerstenberg SI, Loeffler D, Steine MN, Facciotti D (2005) A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol 23(1):75–81. doi:10.1038/nbt1043
70. Slade AJ, Knauf VC (2005) TILLING moves beyond functional genomics into crop improvement. Transgenic Res 14(2):109–115
71. Smyth SJ (2016) Canadian regulatory perspectives on genome engineered crops. GM Crops Food: 0. doi:10.1080/21645698.2016.1257468
72. Soyk S, Muller NA, Park SJ, Schmalenbach I, Jiang K, Hayama R, Zhang L, Van Eck J, Jimenez-Gomez JM, Lippman ZB (2016) Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato. Nat Genet. doi:10.1038/ng.3733
73. Spillane C, Swanson T (2002) Agricultural biotechnology and developing countries: proprietary knowledge and diffusion of benefits. Biotechnology, agriculture and the developing world: The distributional implications of technological change:67–134
74. Sprink T, Eriksson D, Schiemann J, Hartung F (2016a) Regulatory hurdles for genome editing: process- vs. product-based approaches in different regulatory contexts. Plant Cell Rep 35(7):1493–1506. doi:10.1007/s00299-016-1990-2
75. Sprink T, Eriksson D, Schiemann J, Hartung F (2016b) Regulatory hurdles for genome editing: process-vs. product-based approaches in different regulatory contexts. Plant cell reports: 1–14
76. Straubeta A, Lahaye T (2013) Zinc fingers, TAL effectors, or Cas9-based DNA binding proteins: what’s best for targeting desired genome loci? Mol Plant 6(5):1384–1387. doi:10.1093/mp/sst075
77. Sulpice R, McKeown PC (2015) Moving towards a comprehensive map of central plant metabolism. Annual review of plant biology 66 (1)
78. Svitashev S, Young JK, Schwartz C, Gao H, Falco SC, Cigan AM (2015) Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol 169(2):931–945. doi:10.1104/pp.15.00793
79. Tang H, Sezen U, Paterson AH (2010) Domestication and plant genomes. Curr Opin Plant Biol 13(2):160–166. doi:10.1016/j.pbi.2009.10.008
80. Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Young K, Taylor NE, Henikoff JG, Comai L, Henikoff S (2003) Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res 13(3):524–530. doi:10.1101/gr.977903
81. Triques K, Sturbois B, Gallais S, Dalmais M, Chauvin S, Clepet C, Aubourg S, Rameau C, Caboche M, Bendahmane A (2007a) Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea. The Plant journal: for cell and molecular biology 51(6):1116–1125. doi:10.1111/j.1365-313X.2007.03201.x
82. Triques K, Sturbois B, Gallais S, Dalmais M, Chauvin S, Clepet C, Aubourg S, Rameau C, Caboche M, Bendahmane A (2007b) Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea. Plant J 51(6):1116–1125. doi:10.1111/j.1365-313X.2007.03201.x
83. Tsai H, Howell T, Nitcher R, Missirian V, Watson B, Ngo KJ, Lieberman M, Fass J, Uauy C, Tran RK, Khan AA, Filkov V, Tai TH, Dubcovsky J, Comai L (2011) Discovery of rare mutations in populations: TILLING by sequencing. Plant Physiol 156(3):1257–1268. doi:10.1104/pp.110.169748
84. Tsai H, Missirian V, Ngo KJ, Tran RK, Chan SR, Sundaresan V, Comai L (2013) Production of a high-efficiency TILLING population through polyploidization. Plant Physiol 161(4):1604–1614. doi:10.1104/pp.112.213256
85. Uluisik S, Chapman NH, Smith R, Poole M, Adams G, Gillis RB, Besong TM, Sheldon J, Stiegelmeyer S, Perez L, Samsulrizal N, Wang D, Fisk ID, Yang N, Baxter C, Rickett D, Fray R, Blanco-Ulate B, Powell AL, Harding SE, Craigon J, Rose JK, Fich EA, Sun L, Domozych DS, Fraser PD, Tucker GA, Grierson D, Seymour GB (2016) Corrigendum: genetic improvement of tomato by targeted control of fruit softening. Nat Biotechnol 34(10):1072. doi:10.1038/nbt1016-1072d
86. Upadhyay SK, Kumar J, Alok A, Tuli R (2013) RNA-guided genome editing for target gene mutations in wheat. G3: Genes| Genomes| Genetics 3(12):2233–2238
87. van de Wiel C, Lotz L, de Bakker H, Smulders M (2016) Intellectual property rights and native traits in plant breeding. UR Plant Breeding, Wageningen
88. Van Hintum TJL, Brown AHD, Spillane C, Hodgkin T Core collections of Plant Genetic Resources. 2000 In: IPGRI technical bulletin, IPGRI, Rome. pp 1–48
89. van Nimwegen KJ, van Soest RA, Veltman JA, Nelen MR, van der Wilt GJ, Vissers LE, Grutters JP (2016) Is the $1000 genome as near as we think? A cost analysis of next-generation sequencing. Clin Chem. doi:10.1373/clinchem.2016.258632
90. Vicente-Dolera N, Troadec C, Moya M, del Rio-Celestino M, Pomares-Viciana T, Bendahmane A, Pico B, Roman B, Gomez P (2014) First TILLING platform in Cucurbita pepo: a new mutant resource for gene function and crop improvement. PLoS One 9(11):e112743. doi:10.1371/journal.pone.0112743
91. Visendi P, Batley J, Edwards D (2013) Next generation characterisation of cereal genomes for marker discovery. Biology 2(4):1357–1377
92. Voytas DF, Gao C (2014) Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol 12(6):e1001877
93. Waltz E (2016) Gene-edited CRISPR mushroom escapes US regulation. Nature 532(7599):293. doi:10.1038/nature.2016.19754
94. Wang L, Wang L, Tan Q, Fan Q, Zhu H, Hong Z, Zhang Z, Duanmu D (2016) Efficient inactivation of symbiotic nitrogen fixation related genes in Lotus Japonicus using CRISPR-Cas9. Front Plant Sci 7:1333. doi:10.3389/fpls.2016.01333
95. Wang T, Uauy C, Till B, Liu CM (2010) TILLING and associated technologies. J Integr Plant Biol 52(11):1027–1030
96. Wang TL, Uauy C, Robson F, Till B (2012) TILLING in extremis. Plant Biotechnol J 10(7):761–772. doi:10.1111/j.1467-7652.2012.00708.x
97. Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu J-L (2014a) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotech 32(9):947–951. doi:10.1038/nbt.2969http://www.nature.com/nbt/journal/v32/n9/abs/nbt.2969.html-supplementary-information
98. Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu JL (2014b) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32(9):947–951. doi:10.1038/nbt.2969
99. Wang Z-P, Xing H-L, Dong L, Zhang H-Y, Han C-Y, Wang X-C, Chen Q-J (2015) Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol 16(1):1
100. Warschefsky E, Penmetsa RV, Cook DR, von Wettberg EJ (2014) Back to the wilds: tapping evolutionary adaptations for resilient crops through systematic hybridization with crop wild relatives. Am J Bot 101(10):1791–1800. doi:10.3732/ajb.1400116
101. Weil CF (2009) TILLING in grass species. Plant Physiol 149(1):158–164. doi:10.1104/pp.108.128785
102. Wolt JD, Wang K, Yang B (2015) The regulatory status of genome-edited crops. Plant Biotechnol J. doi:10.1111/pbi.12444
103. Zhou H, He M, Li J, Chen L, Huang Z, Zheng S, Zhu L, Ni E, Jiang D, Zhao B, Zhuang C (2016) Development of commercial thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/Cas9-mediated TMS5 editing system. Sci Rep 6:37395. doi:10.1038/srep37395
104. Zhu C, Bortesi L, Baysal C, Twyman RM, Fischer R, Capell T, Schillberg S, Christou P (2016) Characteristics of genome editing mutations in cereal crops. Trends Plant Sci. doi:10.1016/j.tplants.2016.08.009